Proteolytic cleavage of surface proteins enhances susceptibility of lymphocytes to invasion by Theileria parva sporozoites

European Journal of Cell Biology 76, 125-132 (1998, June) . © Gustav Fischer Verlag· Jena

125

Proteolytic cleavage of surface proteins enhances susceptibility of lymphocytes to invasion by Theileria parva sporozoites Josef Syfrig, Clive Wells, Claudia Daubenberger, Antony J. Musoke, J. Naessens 1) International Livestock Research Institute, Nairobi/Kenya Received May 19, 1997 Accepted February 10, 1998

Theileria parva - sporozoite - MHC class 1- CD45R protease

Introduction

A flow cytometric method using anti-parasite antibodies was developed to measure binding of Theileria parva sporozoites to the target bovine lymphocyte membrane. Parasite-host cell interactions could be inhibited by monoclonal antibodies to bovine MHC class I and partially by one of two antibodies to BoCD45R. Proteolysis of the lymphocyte surface removed CD45R but not MHC class I determinants, and enhanced sporozoite binding. These observations support the hypothesis that CD45R and CD45R antibodies may nonspecifically prevent close approximation between sporozoites and lymphocytes. Interestingly, under normal conditions, sporozoites of T. parva did not attach to lymphocytes from goats, but did so when the cells were treated with the protease, suggesting that receptor(s) for T. parva sporozoites might be present on caprine cells but are not easily accessible. These and other results indicate that proteases may be involved in binding and entry of T. parva sporozoites. Electron microscopy revealed that the process of binding and entry of sporozoites into protease-treated goat lymphocytes was very similar to that of the bovine cells. However, schizonts did not develop and lymphocyte proliferation was not induced, indicating that cell entry by sporozoites and cellular transformation are separate processes.

Theileria parva, a tick-borne, obligate intracellular protozoan parasite of cattle, causes East Coast fever, an acute and often fatal lymphoproliferative disease of great economic importance in Eastern, Central and Southern Africa [15]. The mammalian-infective sporozoites of T parva develop within the tick salivary gland and are inoculated into the bovine host during feeding. The sporozoite enters T and B lymphocytes [1] and lyses within minutes the surrounding host cell membrane, thus avoiding lysosomal fusion and destruction. Inside, the parasite develops into a multinucleate body or schizont and induces the host cell to undergo blastogenesis and proliferation. The infected lymphocytes are considered to be transformed because they can be cultivated indefinitely in vitro [5], exhibit surface phenotype alterations [1, 18] and are tumorigenic in athymic [11] or immunodeficient [10] mice. The mechanisms of the T parva sporozoite-lymphocyte interaction are not only of relevance for this model of cell transformation, but also for developing an anti-sporozoite vaccine or a therapeutic drug. Using in vitro neutralization of infectivity of sporozoites by monitoring schizont development by light microscopy, it has been found that antibodies to a sporozoite surface molecule, p67, can prevent parasite invasion of the host cell [8, 16]. However, the in vitro neutralization of infectivity method cannot distinguish between binding and downstream processes, such as entry, schizogony and transformation. Fawcett et al. [9] and Shaw et al. [23] showed that after the initial interaction, the sporozoite and host cell membrane form a more extensive area of attachment followed by an intimate contact leaving no discern able gap between the two membranes and a progressive circumferential "zippering" of the two membranes resulting in the entry of the sporozoite. Shaw et al. [23] demonstrated further, that initial binding can occur at 0-2°C, but that all subsequent steps are temperature dependent. Antibodies against MHC class I could inhibit binding and entry of sporozoitcs into the host cell [23, 22] as analyzed by EM (electron microscopy). Since the binding could occur at 4°C, cocapping of MHC class I-associated

Abbreviations: BSA Bovine serum albumin. - EM Electron microscopy. - FITC Fluorescein isothiocyanate. - MAb Monoclonal antibody. - PBL Peripheral blood lymphocytes. - TSGE Tick salivary gland extract.

I) Dr. Jan Naessens, International Livestock Research Institute (ILRI), P. O. Box 30709, Nairobi/Kenya.

·· ..IRI

126 1. Syfrig, C. Wells, C. Doubenberger et 01. molecules could be ruled out [13]. Sporozoite entry was shown to be proportional to the level of surface expression of MHC class I in lymphocytes [22]. Lymphocytes are the predominant target cells of T. parva sporozoites but are not the only cells that express MHC class L The tropism for lymphocytes must therefore have another basis. The present study was undertaken to analyze further the role of MHC class I and other membrane molecules in the process of sporozoite binding and entry. In addition, the influence of proteolytic cleavage of host cell surface proteins on this process was investigated.

Unbound sporozoites were removed by centrifugation (400g, 1.5 min) and flicking the supernatant of the plate. After gently vortexing the plate, 0.2 % gelatin (Sigma Chemical Co.) in PBS was added to each well. This step was repeated once and PBL were incubated for 15 min in 0.2 % gelatin in PBS. PBL were washed and incubated with MAb 23F (IgG3) or AR22.7 (IgG1) in 0.2 % gelatin in PBS for 30 min. After two washes, the cells were incubated with 100 fll per well of FITC (fluorescein)-labeled antimouse IgGl or FITC-labeled anti-mouse IgG3 (Southern Biotechnology Associates Inc., Birmingham, AL, USA) in 0.2 % gelatin in PBS and washed once more. The cells were then fixed with 2 % formaldehyde (Merck) in PBS and stored at 4°C for up to one week before flow cytometry analysis (FACSCAN, Becton Dickinson, Mountain View, CA). Control samples with uninfected TSGE and without TSGE were included in each experiment and treated under identical conditions.

Analysis of flow cytometry data

Materials and methods Parasites

Rhipicephalus appendiculatus (Muguga stock) ticks were infected as nymphs by feeding on experimentally infected cattle, after which the engorged nymphs were maintained at 23 to 25°C and 80 % relative humidity and allowed to moult to the adult stage. Tick salivary gland extract (TSGE) was prepared by grinding up the dissected salivary glands from infected or uninfected ticks, fed for 4 days on a rabbit, as described previously [6, 23]. Debris was removed by centrifugation (31Og for 7 min) and the supernatant containing the sporozoites saved.

Cell preparations

Peripheral blood lymphocytes (PBL) were obtained from bovine, caprine or human blood as described previously [18]. Blood was collected in Alsever's solution or heparin, and PBL were isolated by centrifugation on Ficoll Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden). The PBL were washed three times in Alsever's solution and resuspended in RPMI 1640 culture medium containing 10 mM Hepes (Gibco Laboratories, Paisley, Scotland).

Monoclonal antibodies

Three thousand signals were acquired and the results were represented as dot plots. To calculate the percentage of positive cells, a threshold was set on the fluorescence signal, such that control samples without sporozoites would have close to 0 % fluorescent positive cells. This threshold was kept the same for all samples of an experiment and the percentage of fluorescent positive cells was determined as the percentage of cells above the threshold. The percentage of sporozoite-binding cells (or positive cells) was obtained by subtracting the values of the samples with uninfected TSGE from the values of the corresponding samples with infected TSGE. Percentage inhibition of sporozoite binding by a MAb was then calculated as the percentage of positive cells of the sample without MAb minus the percentage of positive cells of the sample containing the inhibitory MAb, divided by the percentage positive cells of the sample without MAb, and this figure multiplied by 100. There was a substantial variation in the percentage of fluorescent positive cells from experiment to experiment (ranging from 13 to 81 percent). Shaw et al. [23] reported an infection index at 4°C of 22 % using electron microscopy, with the possibility that this figure was too low because, potentially, one section could not show all bound sporozoites on a cell. Their infection index at 37°C varied from 27 to 39 %. The reasons for the variations in our results could be manifold: batch to batch variations of the infectivity of sporozoites, varying percentages of lymphocytes in PBL preparations, variations in the ratio of tar-

MAb 23F (isotype IgG3) was produced against infected TSGE and inhibits sporozoite entry in the neutralization of infectivity of T. parva sporozoites assay. Another MAb AR22.7 (IgG1) was produced against recombinant p67 sporozoite protein (Musoke, unpublished data). Other antibodies used in these studies include IL-A19 (IgG2a) and IL-A88 (IgG2a) both of which react with nonpolymorphic determinants of bovine MHC class I molecules [4]. MAb W6/32 (IgG2a) and B1G6 (IgG2b), which recognize epitopes on all HLA-A, Band C heavy chains and human beta-2-microglobulin, respectively, crossreact with the homologous monomorphic determinants on bovine MHC class I [3,4]. All other MAb tested for inhibition were obtained from participants of the 2nd workshop on ruminant leukocyte antigens [17] or produced at I.L.R.I., and selected as surface markers of bovine leukocytes.

Sporozoite binding assay

All steps were performed on an ice slurry at 4°C in order to prevent sporozoites from entering the PBL and escaping detection [23]. Fifty thousand PBL in 50 fll RPMI 1640 culture medium with 10 mM Hepes buffer (Gibco Laboratories) were incubated in a well of a round bottom 96-well microtiter plate (Costar, Cambridge, MA, USA) together with 50 fll of infected or uninfected TSGE. Preliminary experiments had shown that 150 infected acini produced a saturating amount of sporozoites per 5 x 104 bovine PBL (result not shown), and this was used in all subsequent experiments. The plate was incubated for 1 h and vortexed gently after 30 min. For inhibition studies, PBL were incubated with MAb for 15 min on ice before addition ofTSGE. Care was taken that the MAb did not have the same isotype as the antisporozoite MAb used to reveal the bound parasites.

Fig. 1. Differential interference contrast (a, b) and fluorescence (c, d) light micrographs of bovine PBL incubated with infected TSGE and subsequently with MAb 23F (a, c) or MAb AR22.7 (b, d) followed by a FITC-conjugated secondary antibody. Sporozoites attached to lymphocytes appear in the fluorescence images. The surface of lymphocytes remains dark. Bar is 10 flm.

Protease enhances lymphocyte susceptibility to T. parva

get cells and variations in the exact dilution of antibodies used for detection. Since we examined our data always relative to each other in one experiment, these variations are unlikely to influence our interpretations.

Direct binding of MAb to PBL measured by flow cytometry

These experiments were performed in the same microtiter plate and with identical proportions of PBL and antibodies as for the inhibition studies of sporozoite binding. A FlTC-labeled goat anti-mouse IgG antibody (Amersham International pic) was used as secondary antibody and the mean value of the fluorescence of all cells in one well was determined after flow cytometry as previously described [19].

Light microscopy For light microscopy, samples were processed as for analysis with the flow cytometer with double concentrations of primary and secondary antibodies. After fixation, the PBL in solution were centrifuged (50g, 6 min) onto glass slides in a Cytospin 2 centrifuge (Shandon, Southern Products Ltd, Runcom UK). The glass slides were dried and coverslips were mounted with Citifluor (Agar Scientific, Stanstead, UK). Samples were examined in a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany).

Results Detection of sporozoite binding by flow cytometry A fast and quantitative assay was developed in order to analyze T parva sporozoite binding to PBL. TSGE and PBL were incubated together at 4 DC in order to allow only attachment of sporozoites, but not their entry [23]. Unbound sporozoites were washed away and the sporozoites attached to the cells were visualized by indirect immunofluorescent staining with one of two MAbs against the sporozoite p67 surface antigen. In light micrographs, sporozoites bound to PBL appear fluorescent while no fluorescence is emitted from the surface of the PBL (Fig. 1). To accelerate data collection and to obtain results from a high number of cells, samples were analyzed by flow cytometry.

IL·A19

Limited digestion of PBL surface proteins with proteinase K One million cells per ml in RPM I 1640 with 10 mM Hepes buffer were added to the same volume of freshly prepared 0.5 mg proteinase K (Serva, Heidelberg, Germany) per ml in RPMI 1640 with lOmM Hepes buffer. This mixture was incubated at 37 DC for 2 h with occasional inversion of the tube to resuspend the cells. Subsequently, the cells were briefly washed twice with 0.2 % gelatin (or 10 % fetal calf serum in experiments for analysis of the entry process) in RPMI 1640 with 10 mM Hepes buffer and incubated with TSGE. Viability of cells was at this stage still higher than 50 % .

Electron microscopy Approximately one million bovine or caprine lymphocytes, either treated with proteinase K or untreated, in 200 [.II RPM I 1640 with 10 mM Hepes buffer were added to 250 [.II TSGE containing 833 infected acini in the same buffer and incubated for 30 min at 37 DC. Samples were taken for examination of sporozoite infectivity and internalization. For following up the development of sporozoites in host cells, PBL-sporozoite mixtures were washed once after 30 minute incubation with RPMI 1640 culture medium containing 10 mM Hepes buffer and 10 % heat-inactivated fetal calf serum (Gibco Laboratories) and incubated in the same medium at 37 DC and at 5 % CO 2 . After different incubation times, suspensions of these cells were fixed by adding equal volumes of 4 % glutaraldehyde in 0.1 M sodium cacodylate buffer. Samples were processed by standard techniques and embedded in an Epon-Araldite mixture. Ultrathin sections (60 nm thickness) were collected onto copper grids, counterstained with Reynolds lead citrate and examined by electron microscopy (EM lOA, Zeiss, Oberkochen, Germany). The results of the EM analysis were quantified as described [23]. Briefly, the infection index is defined as the percentage of host cells showing either surface-bound or fully internalized sporozoites in one section of 300 to 500 cells. On the same section, the sporozoite internalization index was determined, which is defined as the percentage of cell-associated sporozoites that are fully internalized within an infected cell.

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Fig. 2. Four MAbs against MHC class I were tested for their inhibitory capacity on sporozoite binding to bovine PBL measured by flow cytometry. Inhibition was compared with direct binding of the MAbs to PBL. IL-AI9 and W6/32 inhibit sporozoite binding by 50 % close to the concentration for their half maximal binding to PBL. IL-A88 and B 1G6 inhibit sporozoite binding by 50 % only at much higher concentrations than necessary for their half maximal binding to PBL. See Materials and methods for details of analysis.

128 J. Syfrig, C. Wells, C. Doubenberger et 01.

Differences in blocking of sporozoite binding by anti-MHC class I Four MAbs against MHC class I or ~2-microglobulin were tested for their capacity to inhibit sporozoite binding to bovine PBL (Fig. 2). To that end PBL were preincubated with a wide range of concentrations of each MAb before addition of infected or uninfected TSGE. The four MAbs were also analyzed by flow cytometry for their binding to PBL over the same range of concentrations. In this way, we could directly compare the relative capacity of each MAb to block sporozoite binding with its capacity to bind the MHC class I epitope. From the graphs presented in Fig. 2, it is obvious that the binding curves (open circles) of IL-A19 and IL-A88 have the same slope and that the two MAbs bind with similar affinity to MHC class I on the cell surface. However, IL-A19 has a much higher efficiency in blocking sporozoite binding. The concentration of IL-A19 for half maximal binding is about 4.7 x W- 10 M and that of IL-A88 is slightly lower at 1 x 10-10 M. However, their relative concentrations to obtain 50 % inhibition of sporozoite binding are very different: 4.2 x 10-9 and 5.3 x 10-8 M, respectively. This means that slightly less IL-A88 was needed to obtain the same binding as IL-A19, while almost 10 times more was needed to obtain the same inhibition of sporozoite binding. The slope of the binding curves for MAbs W6/32 and B1G6 are more shallow than the previous two Mabs, suggesting a weaker affinity for MHC class I, probably because they were raised against human MHC. Higher concentrations were needed to obtain half maximum binding values: 1.6 x 10-9 M for B1G6 and 5 X 10-9 M forW6/32. Yet, the capacity ofW6/32 to inhibit sporozoite binding is much higher than that of B1G6. The concentrations needed for 50 % sporozoite inhibition were very different: 2.6 x W-7 for B1G6 and 2.1 x 10-8 for W6/32. While 3 times less B1G6 than W6/32 was needed to observe the same binding, 10 times more was needed for the same sporozoite inhibition. From this it is evident that IL-A19 and W6/32 were more effective in preventing binding of sporozoites to bovine PBL than IL-A88 and BIG6. The effectiveness of the four MAbs to inhibit sporozoite binding did not correlate with their affinities for their epitopes on bovine PBL.

Inhibition of sporozoite binding by MAbs against leukocyte antigens To investigate whether molecules other than MHC class I were involved in sporozoite binding to lymphocytes, we screened a panel of MAbs against bovine lymphocyte antigens for inhibition of sporozoite binding (Table I). In a first experiment 42 % of PBL bound sporozoites. An anti-MHC class I MAb, ILA19, strongly inhibited sporozoite binding, while anti-bovine CD45R MAb CC31 induced a partial inhibition. To avoid the possibility of a contaminating antibody, the CC31 hybridoma was recloned, fresh antibody produced and the experiment repeated (experiment 2, Table I). In this experiment, a higher number of lymphocytes bound sporozoites (91 %), and again the anti-MHC class I inhibited very efficiently, while CC31 inhibited partially. None of the other MAbs investigated (including two additional MAbs against bovine CD45 or CD45R) showed a similar inhibition of sporozoite binding to PBL.

Tab. I. Screening of MAbs against leukocyte antigens for inhibition of sporozoite binding. mAB

Isotype

Specificity

Exp. 1 Exp.2 % inhibition

Il-A19 2E8 CC31 CC103 CC76

G2a G2b G1 G1 G1

MHC class I CD45 CD45R CD45R CD45R

67 0 55 9 not done

98 not done 33 not done 4

Bovine PBl were mixed with MAbs prior to incubation with infected TSGE. Bound T. parva sporozoites and percentage of inhibition were estimated as described in Materials and methods. In the first experiment 42.3 % of PBL without MAb bound sporozoites and in the second 90.8 %. The following MAbs against various bovine leukocyte antigens (specificities in brackets) were also tested in the same experiments and did not significantly inhibit: 20-27 (CD1w2), CC20 (CD1w2), CC90(CD1w2), CC122 (CD1w2) CC43 (CD1w3), CC118 (CD1w3), Il-A87 (CD11 a/18), IL-A99 (CD11 a), IL-A 130 (CD11 b), Il-A 111 (CD25), IL-A 112 (CD44), IL-A118 (CD44), Il-A136 (CD44), Il-A77 (CD71), IL-A54 (WC5), Il-A53 (WC6), IL-A163 (WC9), Il-A117 (WC11), P10 (activation/PMN), Il-A 162 (pan-leukocyte), IL-A 119 (p90, activation), Il-A 141 (p90, activation), J5 (p1 00, activation), P5 (T cell/monocyte), and IL-A39, Il-A63, IlA80, IL-A122, Il-A142, IL-A147 (the later all unknown non-lineage antigens). Nomenclature and description of bovine leukocyte antigens as in [17].

Effect of proteinase K treatment of PBl on sporozoite binding In order to investigate whether removal of MHC class I and other possible receptors from the surface of PBL influenced sporozoite binding, bovine PBL were treated with proteases before testing their capacity to bind sporozoites. In agreement with previous observations [25], papain was unable to remove MHC class I from bovine PBL to any significant extent (results not shown). Treatment with proteinase K resulted in a small reduction in MHC class I molecules on the surface of bovine PBL (Table II, first two columns) and did not significantly reduce binding of sporozoites (67 % instead of 71 %) (Table II, third column). MAb IL-A19 was still able to partially inhibit binding to treated cells (down to 45 % from 67%). Caprine PBL were used as control cells and were treated and analyzed in the same way as the bovine cells (Table II). Sporozoites showed low affinity for untreated PBL from goat (14 %), but after proteinase K-treatment caprine PBL bound sporozoites in considerable numbers (48 %) although not to

Tab. II. The effect of proteinase K treatment of PBL on sporozoite binding. Source of PBl

Bovine Bovine Caprine Caprine

Prot. K treatment

+ +

Mean fluorescence % PBl binding sporozoites of PBl stained for in the presence of Il-A19

Il-A88

142 118 75 59

124 115 87 77

71 67 14 48

Il-A19

IL-A88

17 45 6 43

59 68 8 27

PBL were treated with proteinase K or culture medium at 3JDC and preincubated with anti-MHC class I MAb where indicated. Sporozoite binding and direct binding of anti-MHC class I MAb were performed at 4°C and detected as described in Materials and methods. Values represent the means of two experiments.

Proteose enhances lymphocyte susceptibility to

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Flowcytom,etric analysis of normal and proteinase K (PK)treated bovine PBL stained with MAb IL-A19 (anti-MHC class I), CC31 and CC76 (both anti-CD45R). Controls were incubated with secondary antibody only. The enzyme digested CD45R, but not MHC class I molecules.

the same extent as bovine PBL (67 %). It is unlikely that this was due to an effect of proteinase K on the sporozoites because the PBL were washed briefly after the treatment and before adding TSGE. Furthermore, sporozoites treated with proteinase K lost their capacity to bind to lymphocytes (results not shown). Both IL-A19 and IL-A88 cross reacted with MHC class I on caprine PBL (Table II). IL-A88 (and less efficiently IL-A19) inhibited sporozoite binding to proteinase K-treated caprine PBL (from 48 % down to 27 %), suggesting that MHC class I was also involved in binding of sporozoites to proteinase K-treated PBL from goat. While removal of MHC class I epitopes from the cell surface with proteinase K was minor, CD45R was efficiently cleaved from all cells, including the epitope of CC31 (Fig. 3).

Effect of proteinase K treatment of PBL on sporozoite entry To compare sporozoite entry and schizont development between caprine and bovine lymphocytes, infected cells were further examined by electron microscopy. The infection and internalization indices were determined according to Shaw et al. [23]. The sporozoites bound to and entered proteinase K-treated caprine PBL in similar numbers (infection index) as proteinase K-treated bovine PBL (Table III). The infection

Tab. III. The effect of proteinase K treatment of PBL on sporozoite invasion. Source of PBL

Bovine Bovine Bovine Bovine Caprine Caprine

Prot. K IncuInfection index (% of PBL) treatbation ment with MAb IL-A19

+ + +

+ +

Sporozoite internalization (% sporozoites)

expo 1

expo 2

expo 1

expo 2

30.8 62.1 2.8 9.4 0 59.4

28.7 53.8 2.7 7.5 0 56.2

38.1 43.6 0 6.4 0 40.9

36.6 42.1 0 6.1 0 40.1

PBL were incubated with infected TSGE for 30 min at 37°C and processed for electron microscopic examination as described in Material and methods. The infection index is described as the percentage of lymphocytes showing either surface-bound or fully internalized sporozoites in one section. Sporozoite internalization is defined as the percentage of cell-associated sporozoites that are fully internalized within an infected lymphocyte. Results are shown from two different experiments.

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index was lower (around 30 % ) for untreated bovine PBL and no sporozoites were found associated with untreated caprine PBL (infection index 0 %). In proteinase K-treated PBL, from goat or cow, infection of one cell with several sporozoites was often observed. This was a much rarer event in untreated PBL from cattle (result not shown). The initial steps of sporozoite entry into proteinase K-treated caprine PBL were identical or very similar to that in untreated or treated bovine PBL (not shown). The establishment of an intimate contact between the sporozoite and lymphocyte membrane and the progressive circumferential apposition of the two membranes (Fig. 4a, b) proceeded exactly as described for bovine lymphocytes [23]. Fully internalized sporozoites were completely encapsulated by the caprine host cell membrane. As in bovine lymphocytes, there was no discern able gap between the tightly apposed membranes of the sporozoite and the host cell (Fig. 4b). Later in the process, the separation of the two apposed membranes appeared (Fig. 4c), and most parasites had proceeded as far as this stage after 30 min of invasion. However, only a few parasites were detected where the separated host cell membrane had dissolved and the microtubule "basket" was seen forming around the sporozoite (Fig. 4d). In bovine lymphocytes infected in parallel many more sporozoites had reached this stage of development. Thus, the process appeared to be slower in PBL from goats than from cattle. Cultured proteinase K-treated caprine PBL infected with T. parva sporozoites showed no clear signs of schizont development after more than 3 days either by electron microscopy or in Giemsa-stained samples (results not shown). Some of the lymphocytes became enlarged with abundant endoplasmic reticulum; others showed an abnormal number of Iysosomes. A few host cells showed signs of progress to a mitotic stage (beginning of spindle formation), but these occasions were rare, incomplete and uncoordinated. The T. parva-infected bovine PBL, in contrast, were heavily proliferating at this stage. No cell lines could be established from proteinase K-treated caprine PBL infected with T. parva, even after cultivation for two months. We also examined the interaction of T. parva sporozoites with proteinase K-treated lymphocytes from human and mouse by electron microscopy. In both species neither binding, nor entry of the parasite into the cells was observed (results not shown).

Discussion We have developed a flow cytometric method using antisporozoite antibodies, to measure binding of Theileria sporozoites to the membrane of the target lymphocyte. This method has the advantages that it is fast, that a large number of samples can be processed under the same conditions and that it produced quantitative data directly. Using this assay, we confirmed previous reports that MAbs to MHC class I could inhibit sporozoite binding [23, 24]. However, our observations showed no correlation between antibody affinity and the capacity to compete with sporozoite binding. The monoclonal antibodies IL-A19 and W6/32 were much more effective at preventing sporozoite binding than MAbs IL-A88 and BIG6. This could be explained by the location of the epitope on the membrane molecule. W6/32 binds an epitope which lies

130 J. Syfrig, C. Wells, C. Doubenberger et 01.

Fig. 4. (A) Sporozoite of T. parva in the process of entering a caprine lymphocyte pretreated with proteinase K. The establishment of an intimate contact between the sporozoite and lymphocyte membrane and the progressive circumferential apposition of the two membranes proceeds exactly as described for bovine lymphocytes [23]. (8) Similarly to bovine lymphocytes , there is no intercellular gap discern able between the tightly apposed membranes of the sporozoite (S) and host cell (HC) . (C) Entered sporozoites are first completely encapsulated

by the caprine host cell membrane in a fashion similar to the bovine lymphocyte. A separation of the two apposed membranes occurs subsequently (arrowheads) . Most parasites had proceeded to this stage after 30 min of invasion. (D) Very occasionally, the separated host cell membrane was seen to be dissolved (arrowheads) and micro tubules were observed forming around the sporozoite (arrows). This process appears to be slower than in bovine lymphocytes infected in parallel.

mainly on the a2 domain and partially on the a3 domain of human MHC class I [27] , while B1G6 binds an epitope on ~T microglobulin [12]. We therefore predict that the binding site for sporozoite ligands lies closer to the a2 and/or the a3 domain of MHC class I. Epitope mapping of IL-A19 and IL-A88 may provide more information. Presence of MHC class I alone is not sufficient for binding of sporozoites [24], since not all MHC class I-expressing cells,

such as granulocytes and fibroblasts, bind sporozoites. One or several other membrane molecules may therefore playa role . A panel of MAbs with specificity for lymphocyte membrane molecules, including CD1 and pan-leukocyte antigens, was assayed for their capacity to interfere with sporozoite binding. Only one MAb with specificity for CD45R inhibited sporozoite binding (although not as strong as MAb against MHC class I) ; asecond CD45R MAb did not inhibit. It is possible that the

Proteose enhances lymphocyte susceptibility to

second MAb detects an epitope which is not in the vicinity of the sporozoite binding site [13]. This observation suggests that CD45R may play a role in the binding process. In cattle, CD45R was shown to be expressed on 100 % of B lymphocytes (CD21 +) and over 50 % of the Tlymphocytes (CD2+), but not on granulocytes and monocytes [2] . Interestingly, removal of surface antigens from bovine and caprine cells by proteinase K enhanced their capacity to bind sporozoites. Proteinase K was not efficient in cleaving MHC class I from the bovine or caprine cell surface, but CD45R and probably many other surface proteins were removed as shown by lack of binding with specific MAbs after treatment . This result does not agree with the results of one experiment that reported a decrease of both sporozoite binding and internalization after proteinase K treatment [25]. However, it is noteworthy that Shaw mentions that this effect is reversible after 30-60 minutes in the absence of enzyme, resulting in a marginal increase in parasite binding over untreated cells. Also , Shaw neutralized proteinase K with PMSF. Previously, this protease inhibitor has been shown to inhibit sporozoite entry [23]. It is possible that residual PMSF in the cell suspension initially inhibited sporozoite attachment. Sporozoites did not bind normal , untreated caprine cells, as expected from previous observations that goat cells do not become infected by incubation with T. parva sporozoites [26]. However, there was strong binding and internalization of parasites in caprine cells after proteinase K treatment. Examination by electron microscopy confirmed that binding and entry of sporozoites in treated bovine and caprine cells was ultrastructurally indistinguishable from binding and entry in untreated bovine lymphocytes . The specificity of this process was further demonstrated by the fact that there was virtually no binding and uptake of tick salivary gland organelles by lymphocytes after proteinase K treatment and that no sporozoites were observed attached to granulocytes or erythrocytes present in the same sample . These observations further suggest that MHC class I may be one partner necessary for sporozoite binding, but that other membrane molecules are involved . Two hypotheses may explain these observations. MAbs to surface antigens may inhibit sporozoite binding by blocking a specific receptor, or by preventing close approximation of cell and sporozoite in a non-specific manner, such as increasing steric hindrance. CD45 is a large molecule [13] that covers about 10 % of the lymphocyte surface and has been thought to obstruct contact between T cells and antigen-presenting cells [7]. Binding of a MAb to CD45R may increase steric hindrance , thereby reducing sporozoite binding, while removal of a part of the CD45R molecule (and other molecules) by protease treatment, may improve accessibility of the host cell surface to sporozoites, thereby enhancing sporozoite binding. Removal of CD45R in goat cells may further expose the receptor(s) for T. parva sporozoites. It is well known that some Theileria species, T. hirci and T. avis , infect goats, but none infect man or mice [28], thus suggesting that Theileria sporozoite receptors are present on goat, but not on human or mouse cells. The other possibility is that CD45R, like MHC class I, is a specific co-receptor. Its expression on lymphoid but not myeloid cells could help explain why binding of T. parva sporozoites is restricted to lymphocytes. However, it becomes more difficult to explain how proteinase K treatment could enhance binding, unless the active site on CD45R was not cleaved off.

T. porva

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Although the initial stages of sporozoite attachment and entry in protease-treated caprine cells were similar as in bovine cells, subsequent developments (the separation of the apposed host cell and parasite membranes and the formation of a microtubule basket) differed between the two species. Our data confirmed that schizont development and cell transformation depend on species-restricted processes after binding and entry of the sporozoite. Our results clearly demonstrate that there is an advantage for T. parva to bind to lymphocytes with at least partially removed surface proteins, implicating the possible involvement of one or more proteases in the process. Shaw et al. [23] showed that PMSF, an inhibitor of serine proteases, inhibited sporozoite binding, while other less specific protease inhibitors had no effect. The observation that susceptibility of bovine lymphocytes to sporozoite invasion increased after incubation with uninfected TSGE [24] , would suggest that protease activity of tick origin may play a role. Iodoacetamide, a general inhibitor of cysteine proteases, did not inhibit binding (data not shown) suggesting that the previously detected T. parva cysteine protease [20] was not involved. There are other examples of parasite-host interactions which depend on the presence of proteases or other enzymes [14, 21]. Isolation of potentially active proteases from infected TSGE would be useful for future studies on the T. parva sporozoite-lymphocyte interactions and development of a treatment for East Coast fever. Acknowledgements. The authors wish to thank James Gachanja and Dismus Lugo for technical help, James Magondu and Peter Mucheru for help with the flow cytometer. This research was partially funded by the Swiss National Science Foundation and the Ciba-Geigy Jubilee Foundation (J. Syfrig) and by the Belgian Government (J. Naessens) . This paper carries ILRI reference number 98008.

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