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The Journal of Immunology, 2000, 164: 2037-2045.
Copyright © 2000 by The American Association of Immunologists

Selection of Babesia bovis-Infected Erythrocytes for Adhesion to Endothelial Cells Coselects for Altered Variant Erythrocyte Surface Antigen Isoforms1

Roberta M. O’Connor and David R. Allred2

Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611.


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Sequestration of Babesia bovis-infected erythrocytes (IRBCs) in the host microvasculature is thought to constitute an important mechanism of immune evasion. Since Ig is considered to be important for protection from disease, an in vitro assay of B. bovis sequestration was used to explore the ability of anti-B. bovis Ig to interfere with IRBC cytoadhesion, and to identify IRBC surface Ags acting as endothelial cell receptors. Bovine infection sera reactive with the IRBC surface inhibited and even reversed the binding of IRBCs to bovine brain capillary endothelial cells (BBECs). This activity is at least partially attributable to serum IgG. IgG isolated from inhibitory serum captured the variant erythrocyte surface ag 1 (VESA1) in surface-specific immunoprecipitations of B. bovis-IRBCs. Selection for the cytoadhesive phenotype concurrently selected for antigenic and structural changes in the VESA1 Ag. In addition, the anti-VESA1 mAb, 4D9.1G1, proved capable of effectively inhibiting and reversing binding of adhesive, mAb-reactive parasites to BBECs, and by immunoelectron microscopy localized VESA1 to the external tips of the IRBC membrane knobs. These data are consistent with a link between antigenic variation and cytoadherence in B. bovis and suggest that the VESA1 Ag acts as an endothelial cell ligand on the B. bovis-IRBC.


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
Babesia bovis is one of many hemoparasites capable of establishing chronic infections of very long duration (1, 2, 3). Although the bovine host can mount an immune response that controls the disease (2), the parasite continues to proliferate in the bloodstream at parasitemias that are often well below the level of microscopic detection (1, 2, 3). The ability of B. bovis to establish asymptomatic, chronic infections suggests that an equilibrium develops between the anti-parasitic immune defenses of the host and the parasite’s immune evasion mechanisms.

The host immune mechanisms that suppress parasite proliferation are poorly understood. Although passive transfer of homologous IgG from hyperimmunized cattle can provide protection from acute disease (4), the mechanisms behind this protection have not been fully investigated. The kinetics of parasite clearance suggested that recognition of infected erythrocytes (IRBCs)3 by serum Abs was important for removal of the parasites from the circulation (4). Ab-dependent cellular cytotoxicity (ADCC) (5, 6), and opsonization and phagocytosis (7), are two mechanisms by which cells of the immune system may eliminate IRBCs. However, serum Abs could also effect the destruction of B. bovis IRBCs by disruption of cytoadherence and IRBC sequestration. Erythrocytes carrying trophozoite and mature meront stages of B. bovis are capable of adhering to capillary endothelial cells in vivo (8, 9, 10). This process results in the concentration of IRBCs in the host microvasculature (8, 9, 11), a behavior referred to as "sequestration." It is thought that sequestration prevents erythrocytes that carry mature parasite stages from being cleared by passage through the spleen, as well as rendering them somewhat inaccessible to other Ab-dependent cellular immune defenses (12). As the IRBC surface is altered structurally (13) and antigenically (3, 14, 15, 16) by the intracellular parasite, reversal of sequestration through Ab recognition of IRBC surface Ags could force IRBCs into the circulation where they might be more rapidly eliminated. In addition, immune clearance of the sequestered IRBCs from the microvasculature could potentially alleviate much of the pathology associated with the acute disease (11). Studies testing these contentions in babesiosis, however, have not been reported.

The B. bovis-IRBC surface Ags that elicit host Abs during infection are antigenically diverse (14, 16), and one of these, the variant erythrocyte surface Ag 1 (VESA1), undergoes clonal antigenic variation within the host (3, 17). Regardless of the mechanism by which Ab effects destruction of the IRBC, variation of the Ag target has the potential to nullify the efficacy of an existing immune response, requiring the host to generate a new response. If antigenic variation of the IRBC surface and IRBC-endothelial cell binding were linked genetically, this would provide the parasite with two benefits: 1) an efficient means of evading the humoral immune response, and 2) increased diversity in its cytoadhesion ligand(s), with the potential to generate molecules capable of binding different endothelial cell receptors. Among hemoparasites, this scenario is not without precedent. Antigenic variation of the Plasmodium falciparum-IRBC surface (18) and in vitro IRBC-cytoadhesive properties (19) both localize to the same molecule, PfEMP1 (20, 21, 22, 23), which has been shown to interact with several endothelial cell receptors (24, 25). To date, the components involved in B. bovis cytoadhesion have not been definitively identified, nor has linkage of antigenic variation and cytoadhesion been reported.

Recently, we developed an in vitro cytoadhesion assay to investigate B. bovis cytoadhesion to endothelial cells (26). Here, we report the use of this assay to test the ability of serum Abs to disrupt the IRBC-endothelial cell interaction, and to begin identification of the IRBC cytoadherence ligand. Results from these experiments strongly suggest that antigenic variation and cytoadhesion are linked in B. bovis and provide the first indication that both properties may be functions of the same molecule.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Bovine sera and mAbs

Several different sources of bovine infection and hyperimmune sera were used in these experiments. Calf B2442 was infected by subinoculation with blood pooled from four calves that had been chronically infected with the MO7 parasite clone. At the same time, in vitro cultures were established with the pooled blood, and B2442 was rechallenged three times with these parasite cultures (14). The C9.1A-I+ clonal line was derived from these cultures. B147 "time-course" sera were collected from a spleen-intact animal at 0, 14, 33, 58, 83, and 105 days postinfection with C9.1A-I+ (3). Splenectomized calves B103 and B106 received blood from calf B147 that was collected either 19 (B103) or 41 (B106) days postinfection (3). Spleen-intact calves B117 and B119 were infected by subinoculation with blood from B103 and B106, 9 and 8 days postinfection, respectively. Rabbit sera were raised against bovine erythrocytes by immunization with erythrocyte ghosts, and against bovine glycophorin by immunization with the purified protein (27). MAb 4D9.1G1, generated by immunization with C9.1A-I+-IRBCs, identifies a surface-exposed, variant epitope on the B. bovis C9.1A-I+ VESA1a polypeptide (17). The negative isotype control mAb HL298, which recognizes an 85-kDa cold acclimation protein of spinach, was a gift from Dr. Charles Guy (University of Florida, Gainesville, FL). MAb Babb35A4, which reacts with the B. bovis merozoite surface Ag, MSA1 (28), was a gift from Dr. Guy Palmer (Washington State University, Pullman, WA).

Parasites

B. bovis parasites were maintained in vitro under microaerophilous conditions, as described previously (3). For clarity, the phenotypes of the parasite lines described in this paper are denoted in superscript as "A+" for adhesive, and "A-" for nonadhesive to bovine brain capillary endothelial cells (BBECs) in vitro, and as "I+" or "I-" for IRBC immunoreactivity or nonreactivity, respectively, with the mAb 4D9.1G1. The derivations of the B. bovis clonal line MO7A-I- and its progeny clone, C9.1A-I+, have been described elsewhere (3, 28). D41A-I- parasite cultures were established from parasites collected from the peripheral circulation of calf B147 at 41 days postinoculation with C9.1A-I+ (3). C9.1A+I-, MO7A+I-, and d41A+I- were parasite populations derived from repeated selection of the nonadherent parental cultures for binding to endothelial cells (26). MO7A+I- was subjected to further selection for mAb 4D9.1G1 reactivity (29) that resulted in the isolation of the nonadherent line, MO7A-I+ (26). The MO7A-I+ population was selected for endothelial cell binding, and again for mAb reactivity to yield the MO7A+I+ population. Two clonal lines were established from this parasite line: CD7A+I+ and CE11A+I- (26).

Endothelial cells

Two sources of BBEC were used in the cytoadhesion assays. The BBEC used in some assays were a gift from Dr. Richard Weiner (University of California, San Fransisco, CA). BBEC were also isolated in our laboratory, essentially as described by Carson and Haudenschild (30). The endothelial cell origin of both BBEC lines was confirmed by uptake of acetylated low density lipoprotein (31), and expression of Von Willebrand factor (26). In the cytoadhesion assays, similar results were obtained regardless of the source of BBECs.

Live-cell immunofluorescence assays (live-cell IFA)

The procedure used to detect the IRBC surface reactivity of bovine sera and mAbs has been described in several publications (3, 14, 17, 32). In live-cell IFAs using rabbit sera as the primary Ab, reactions were amplified with monoclonal anti-rabbit Igs (clone RG-16, Sigma, St. Louis, MO) and the immune complexes detected with FITC-conjugated goat anti-mouse IgG. All IFAs were repeated twice, and each replicate was read by a different individual. Slides were coded and read blindly.

Cytoadhesion assays

This procedure is described in detail elsewhere (26). Briefly, BBECs were seeded in the wells of 24-well plates containing Thermonox coverslips (Nunc Nalge International, Milwaukee, WI). After the cells were established and proliferating in the plates, the medium was removed, and parasite cultures at 2–5% parasitized erythrocytes and 1% packed cell volume in parasite growth medium (40% normal bovine serum (NBS) in medium 199 containing 20 mM 2-tris-(hydroxymethyl)methylaminoethane sulfonic acid (M199/TES)) (33) were added to the endothelial cells. After a 90-min incubation under microaerophilous conditions, with agitation every 15 min, any nonadherent cells were removed by three washes with HBSS. The cells were fixed in methanol and stained with Giemsa stain, and the coverslips were mounted on slides. A 1-cm2 grid divided into nine equal squares was drawn on the coverslip, and the number of IRBCs binding to 16–20 endothelial cells in each of the nine squares was determined. The slides were coded and read blindly to avoid reader bias. Within an assay, each sample was run in duplicate, and each experiment was repeated three times.

To test the ability of sera and mAbs to inhibit or reverse binding, the following modifications were made. For inhibition of cytoadhesion with bovine serum, 50% (v/v) of the normal bovine serum in the parasite growth medium was replaced with experimental serum, resulting in a final concentration of 20% (v/v) experimental serum. This medium was added to the endothelial cells before the parasitized erythrocytes. For reversal of cytoadhesion with sera, IRBCs were allowed to bind to endothelial cells, after which the medium was replaced with medium containing 40% (v/v) experimental serum. Incubation was then continued for another hour before washing and fixation. Assays including mAbs or rabbit sera were performed as described for bovine serum, except that the parasites were added to the BBECs in M199/TES instead of parasite growth medium. For cytoadhesion inhibition, the mAbs were used at 50 µg/ml and the rabbit sera at 5% (v/v) of the total volume. For reversal of cytoadhesion, 100 µg/ml of mAbs and 10% (v/v) rabbit sera were used. Log-normal transformed data from inhibition and reversal assays were analyzed by two-way ANOVA, followed by post hoc Bonferroni t tests to determine significant differences between preimmune and immune samples, and between samples treated with mAbs HL298 and 4D9.1G1.

Serum fractionation

Total IgG was isolated from sera by affinity chromatography over a Protein A/G-agarose column (Pierce, Rockford, IL). Serum Abs were bound to the column in 100 mM Tris (pH 7.5) and eluted with 100 mM glycine-HCl (pH 2.5). Eluted Abs were neutralized with 1 M Tris (pH 8.0), exchanged into PBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM sodium phosphate dibasic, 1.5 mM potassium phosphate monobasic (pH 7.4)) over a Bio-Gel P-6 desalting column (Bio-Rad, Hercules, CA), and concentrated to 0.1x the original volume. The Protein A/G column flow-through was also collected, placed in dialysis tubing (m.w. cut-off of 3500, Pierce), and concentrated osmotically with polyethylene glycol 8000 to 1.0x the serum volume. Residual Tris buffer was removed by dialysis against PBS. This preparation, hereafter referred to as IgG-depleted serum, was used in cytoadhesion inhibition assays in essentially the same manner as whole bovine serum. The serum IgG was added to the assay in parasite growth medium to yield concentrations equivalent to those that were present in unfractionated serum.

Surface-specific and conventional immunoprecipitations

The details of surface-specific immunoprecipitations with bovine serum, and conventional immunoprecipitations with mAbs are described elsewhere (3, 14, 17, 32). L-[35S]methionine-labeled IRBCs were used as the Ag source, since mature bovine erythrocytes fail to metabolically incorporate this label into protein. When surface immunoprecipitations were done using IgG-depleted sera, mouse anti-bovine IgM (clone BM 23, Sigma) was used to bridge between the immune complexes and the protein G-Sepharose. In one experiment, the NP40-insoluble immune complexes were analyzed as described elsewhere (14).

Immunoelectron microscopy

B. bovis C9.1A-I+ and CD7A+I+ IRBCs were washed into M199/TES and incubated for 1 h at 4°C in anti-VESA1a mAb 4D9.1G1 or isotype control HL298 at final concentrations of 0.2 mg/ml. The cells were washed three times in M199/TES containing 1% (v/v) normal goat serum (M199/TES/NGS), at 4°C, with centrifugation at 700 x g between washes, before amplification of the primary Ab reactions with 50 µg/ml rabbit anti-mouse (IgG + IgM) diluted in M199/TES/NGS for 1 h at 4°C. The cells were washed as described above; then immune complexes were localized by incubation with 10 nm gold colloid-conjugated goat anti-rabbit IgG (BB International, Cardiff, England) at 2.5 µg/ml.

After immunolabeling, cells were washed into 0.1 M sodium phosphate buffer (pH 7.4), and fixed with 2.0% (w/v) gluteraldehyde in 0.1 M sodium phosphate (pH 7.4), 4.0% (w/v) sucrose, 2 x 10-5 M CaCl2 (34) for 1 h at room temperature, followed by an overnight incubation at 4°C. The cells were dehydrated in an ascending ethanol series, then infiltrated in 1:1 (v/v) then 3:1 (v/v) Spurr’s resin (Electron Microscopy Sciences, Ft. Washington, PA):ethanol, before embedment in Spurr’s resin. Sections were counterstained with 2% uranyl acetate in 75% ethanol and Reynolds lead citrate (35).


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
Inhibition and reversal of cytoadhesion by bovine immune sera and IgG

Several hyperimmune and infection sera raised against related B. bovis clones, and derivatives of those clones, were screened for surface reactivity with CD7A+I+ and CE11A+I- IRBCs. Two infection sera (B117 and B119) recognized the surface of both CD7A+I+ and CE11A+I- IRBCs (Fig. 1GoA). When included during cytoadhesion, both infection sera significantly inhibited cytoadhesion of IRBCs to BBECs (Fig. 1GoB; p < 0.001 for CD7 inhibited with B119 and B117 sera; p < 0.001 for CE11 inhibited with B119 serum; and p < 0.017 for CE11 inhibited with B117 serum). The antisera were also capable of disrupting CD7A+I+ and CE11A+I- IRBCs that had already adhered to BBEC (Fig. 1GoC; p = 0.001 for both CD7 and CE11 with both immune sera).



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  		FIGURE 1. Inhibition and reversal of B. bovis-IRBC adhesion to BBECs by immune sera. Bovine sera B117 and B119 were reacted with the surface of CD7A+I+ and CE11A+I- IRBCs in live-cell IFAs (A), and tested for the ability to inhibit (B) and reverse (C) adhesion of IRBCs to BBECs. pre, preimmune sera; immune, infection sera. Error bars indicate the SEM for two experiments (A) and three experiments (B and C).

 
Since whole serum was used to inhibit and reverse cytoadhesion, it was important to determine whether this effect was due to anti-IRBC Igs or other components present in the serum. To determine whether anti-B. bovis IgG alone could inhibit cytoadhesion, IgG was isolated from B119 preimmune and infection sera and tested for the ability to inhibit IRBC binding to BBEC (Fig. 2Go). The ability to inhibit CE11A+I- cytoadherence was contained predominantly in the IgG fraction (p = 0.004), although repletion of the IgG-depleted serum with purified IgG resulted in an enhanced reduction in the density of cells bound (Fig. 2Go, p < 0.001). B119 serum fractions lost the ability to inhibit CD7A+I+ cytoadherence to a significant degree, although when recombined these fractions did reduce the numbers of CD7A+I+ IRBCs binding to endothelial cells (Fig. 2Go, p = 0.06). In general, B119 whole serum did not disrupt BBEC binding by CD7A+I+ as effectively as that of CE11A+I- IRBCs. It is possible that activity sufficient to achieve statistically significant inhibition of CD7A+I+ adhesion failed to survive the fractionation process. This scenario is not an unlikely outcome if IgM is relatively more significant to inhibition of CD7A+I+ than is IgG.



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  		FIGURE 2. Inhibition of IRBC adhesion to BBECs in the presence of fractionated serum. IgG isolated from B119 preimmune (pre) and infection (immune) sera was tested for the ability to inhibit cytoadhesion of CD7A+I+ and CE11A+I- IRBCs, along with the IgG-depleted serum (depleted) and the recombined fractions (repleted). Error bars indicate SEM for three experiments.

 
Identification of Ags recognized by fractionated bovine sera

To identify the IRBC-surface Ags recognized by the B119 serum, surface-specific immunoprecipitations were performed using B119 IgG and IgG-depleted sera to precipitate L-[35S]methionine-labeled parasite proteins. Because the IgG-depleted sera could contain IgM reactive with the IRBC surface, mouse anti-bovine IgM was included in these immunoprecipitations to facilitate capture of IgM-Ag immune complexes. Both infection serum fractions specifically precipitated the VESA1 Ag, the size of which varied slightly between CD7A+I+ and CE11A+I- (Fig. 3Go, lanes 1-4, arrowheads). The VESA1 was not captured by fractionated B119 preimmune serum (Fig. 3Go, lanes 5-8), or in precipitations performed with mouse anti-bovine IgM alone (Fig. 3Go, lanes 9 and 10).



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  		FIGURE 3. Surface-specific immunoprecipitations performed on CD7A+I+ and CE11A+I- IRBCs with IgG and IgM from infection serum. Lanes 1, 2, 5, 6, and 9, CD7A+I+ IRBCs; lanes 3, 4, 7, 8, and 10, CE11A+I- IRBCs. Lanes 1 and 3, B119 immune IgG; lanes 2 and 4, B119 immune IgG-depleted serum; lanes 5 and 7, B119 preimmune IgG; lanes 6 and 8, B119 preimmune IgG-depleted serum; lanes 9 and 10, mouse anti-bovine IgM. Arrowheads indicate the VESA1 Ag precipitated by B119 IgG and depleted serum. Lane S contains 14C-m.w. standards.

 
Changes in the antigenicity of the IRBC surface after selection for adhesion

Changes that occur on the IRBC surface generally and on the VESA1 Ag specifically as a result of selection for in vitro cytoadherence were examined using three nonadhesive parental parasite lines and the adhesive progeny lines derived from them. The binding characteristics and mAb reactivity of these parasite populations are shown in Fig. 4GoA and in Fig. 5Go. Because late time point sera did not effectively distinguish between nonselected and selected populations, B147 sera, collected at several time points early in infection, were used in live-cell IFAs to compare surface-reactivity of d41A-I- and d41A+I- IRBCs (Fig. 4Go). As shown previously (3), sera from B147 did not react with d41A-I- parasites at high levels until 58 days postinfection, after which time the percentage of IRBCs reactive with serum Abs climbed to nearly 100%. This pattern of reactivity is expected in infections with organisms capable of antigenically varying surface proteins. In general, Abs present in the circulation do not react with the parasite variant present at that same time point. However, Abs generated later during infection react with variants present at earlier time points. In contrast, d41A+I- IRBCs expressed surface epitopes that reacted primarily with d33 and d105 sera, creating a pattern of reactivity with the time course sera distinctly different from that of d41A-I- IRBCs. Surface reactivity of the time course sera correlated well with the ability to reverse binding of d41A+I- (Fig. 4GoB), further supporting the role of surface-reactive IgG in the disruption of cytoadherence by infection sera.



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  		FIGURE 4. Recognition of the IRBC surface and reversal of in vitro cytoadherence with time-course sera. Erythrocytes infected with the nonadherent d41A-I- parental population and the adherent progeny d41A+I- population (A) were assayed in live-cell IFAs with serum collected from calf B147 at 0, 14, 33, 58, 83, and 105 days postinfection (B). The d41A+I- population was exposed to the sera following in vitro cytoadhesion to assay for the ability of the sera to reverse cytoadhesion (B). Error bars indicate SEM for two experiments.

 


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  		FIGURE 5. The binding phenotypes and mAb surface reactivity of parasite populations selected from the C9.1A-I+ and MO7A-I- clonal lines. The mean binding densities are derived from the results of triplicate experiments; error bars indicate SEM. Results from representative live-cell IFAs are given to demonstrate surface reactivity with the mAb 4D9.1G1. Within each group, the order of derivation of individual parasite lines is shown from left to right.

 
Different isoforms of the VESA1 are expressed on IRBC selected for cytoadhesion

To examine changes in specific IRBC surface Ags that might have resulted from the selection of C9.1A-I+ for cytoadherence (Fig. 5Go), surface-specific immunoprecipitations with preimmune and hyperimmune B2442 sera were performed. Both subunits of the VESA1 doublet precipitated from the cytoadherent C9.1A+I- line were different in apparent mass, based on SDS-PAGE analysis, from those precipitated from the parental C9.1A-I+ clonal population (Fig. 6GoA, lanes 1 and 2, arrowheads). To look for changes in other putative IRBC surface Ags, Ag-Ab complexes in the NP40-insoluble fraction were also precipitated (Fig. 6GoA, lanes 3 and 4). No apparent size polymorphisms were observed in other precipitated Ags (Fig. 6GoA, lanes 1-4). Conventional immunoprecipitations of C9.1A-I+ and C9.1A+I- with mAb 4D9.1G1 confirmed that selection for cytoadherence also resulted in the loss of the epitope recognized by mAb 4D9.1G1 (Fig. 6GoA, lanes 9 and 10). Precipitation of MSA-1 (28) from these two populations demonstrated that the immunoprecipitations were done on equivalent amounts of parasite materials, and also that this merozoite surface Ag was apparently unaffected by selection for cytoadhesion (Fig. 6GoA, lanes 11 and 12).



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  		FIGURE 6. Structural and antigenic changes of the VESA1 Ag following selection for endothelial cell binding. A, Surface and conventional immunoprecipitations of L-[35S]methionine-labeled C9.1A-I+ and C9.1A+I- parasite proteins. Polypeptides precipitated from C9.1A-I+ are in lanes 1, 3, 5, 7, 9, 11, and 13, and from C9.1A+I- in lanes 2, 4, 6, 8, 10, 12, and 14. Lanes 1–8, surface immunoprecipitations with B2442 hyperimmune (lanes 1–4) and preimmune sera (lanes 5–8). Lanes 1, 2, 5, and 6, NP40 soluble fractions; lanes 3, 4, 7, and 8, urea soluble components. The VESA1 Ags precipitated from C9.1A-I+ and C9.1A+I- are indicated with arrowheads (lanes 1 and 2). Lanes 9–14, conventional immunoprecipitations with mAbs 4D9.1G1 (lanes 9 and 10), anti-MSA1 mAb Babb35A4 (lanes 11 and 12) and isotype control HL298 (lanes 13 and 14). B, Conventional immunoprecipitations of L-[35S]methionine-labeled parasite materials from MO7-derived parasite lines. Polypeptides precipitated from MO7A-I- are in lanes 1, 7, and 13; MO7A+I-, lanes 2, 8, and 14; MO7A-I+, lanes 3, 9, and 15; MO7A+I+, lanes 4, 10, and 16; CD7A+I+, lanes 5, 11, and 17; and CE11A+I-, lanes 6, 12, and 18. Polypeptides were precipitated with mAbs HL298 (lanes 1–6), 4D9.1G1 (lanes 7–12) or Babb35A4 (lanes 13–18). Changes in the size of the VESA1 doublet are indicated with arrowheads. Lanes marked S in both panels contain 14C-labeled m.w. markers.

 
To ask whether this apparent linkage of adhesive and immunoreactive phenotypes was reproducible, a second parasite line was similarly evaluated. Parasite populations selected from the MO7 clonal line for cytoadhesive phenotype and mAb 4D9.1G1 reactivity (26) were immunoprecipitated with this same mAb. The effects of these selections on the phenotypes of these four populations are shown in Figs. 5Go and 6GoB. Neither MO7A-I- nor MO7A+I- was reactive with mAb 4D9.1G1 (Figs. 5Go and 6GoB, lanes 7 and 8). When MO7A+I- was selected for reactivity with mAb 4D9.1G1, the density of IRBCs binding to BBECs dropped to less than 2 IRBCs per 100 endothelial cells (MO7A-I+, Fig. 5Go). However, a subpopulation that both expressed the 4D9.1G1 epitope, and retained the cytoadhesive phenotype (MO7A+I+, Fig. 5Go), could be recovered. This population expressed a VESA1 isoform that was structurally different from the MO7A-I+ population from which it was derived (Fig. 6GoB, lanes 9 and 10, arrowheads). MAb 4D9.1G1 precipitated an apparently identically sized VESA1 from MO7A+I+ and CD7A+I+, the clonal population derived from MO7A+I+ (Fig. 6GoB, lane 11), but did not precipitate VESA1 from CE11A+I- IRBCs, as expected (Fig. 6GoB, lane 12). Precipitation of MSA1 with mAb Babb35A4 again demonstrated that these extensive selections for different surface Ag and cytoadherence phenotypes had no apparent effect on the merozoite Ag (Fig. 6GoB, lanes 13-18). Negative control immunoprecipitations with the mAb HL298 are shown in lanes 1-6.

Localization of VESA1 to the knobs

The preceding data suggested that VESA1 and cytoadhesive phenotypes are linked. Therefore, the mAb 4D9.1G1 was used to determine where this Ag localized relative to the knob-like structures previously shown to be the point of attachment between IRBCs and endothelial cells (26). Preembedment immunostaining with this Ab demonstrated that the VESA1 Ag was expressed on the tips of the knobby protrusions of the plasma membrane on both CD7A+I+ IRBCs (Fig. 7GoA) and on C9.1A-I+ (not shown). Isotype control mAb HL298 did not label the surface of either parasite line (Fig. 7GoB, and data not shown).



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  		FIGURE 7. Immunolocalization of the VESA1 Ag on CD7A+I+ IRBCs with mAb 4D9.1G1. A and B, CD7A+I+ IRBCs labeled with mAb 4D9.1G1 (A) and isotype control mAb HL298 (B). Identical results were obtained with C9.1A-I+ IRBCs. Arrows indicate the knobs enlarged in the inset in A. P, parasite; PE, parasitized erythrocyte. Bars indicate 0.5 µm in A and B, and 0.1 µm in the A inset.

 
Inhibition and reversal of CD7A+I+ adhesion with mAb 4D9.1G1

Because of the apparent linkage of cytoadhesion and VESA1 Ag phenotypes, and the location of VESA1 to the tips of the IRBC knob structures, the possibility was raised that the VESA1 Ag might act as a cytoadherence ligand for B. bovis-IRBCs. This hypothesis was tested using the cytoadhesive parasite populations CD7A+I+ and CE11A+I-. Cytoadhesion assays were conducted in the presence of anti-VESA1 mAb 4D9.1G1 or the isotype control mAb, HL298 (Fig. 8GoB). The density of CD7A+I+ IRBCs adhering to BBECs was dramatically reduced in the presence of the mAb 4D9.1G1 (Fig. 8GoB, p < 0.001). In contrast, cytoadhesion of the mAb 4D9.1G1 nonreactive clonal line, CE11A+I- (Fig. 8GoA) was unaffected by the presence of this mAb (Fig. 8GoB). Isotype control mAb HL298 had no affect on cytoadhesion of either parasite line. Because inhibition with the mAb could be due to steric effects, rabbit sera raised against bovine RBC ghosts or bovine glycophorin were also tested for their ability to inhibit cytoadhesion. Although both antisera reacted with the surface of all intact RBCs, whether infected (Fig. 8GoA) or uninfected (data not shown), only the anti-RBC serum appeared to interfere with adhesion of CD7A+I+ and CE11A+I- IRBCs to BBECs (Fig. 8GoB). This inhibition was not consistent enough between repeated assays to achieve statistical significance (p = 0.182 for CD7; p = 0.227 for CE11).



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  		FIGURE 8. Inhibition and reversal of CD7A+I+ IRBC adhesion to endothelial cells by mAb 4D9.1G1. MAb 4D9.1G1 and rabbit sera raised against bovine RBC ghosts (anti-RBC) or bovine glycophorin (anti-GPH) were tested for surface reactivity with CD7A+I+ and CE11A+I- IRBCs (A), and for the ability to inhibit cytoadhesion of CD7A+I+ and CE11A+I- IRBCs to BBECs (B). C, MAb 4D9.1G1 and rabbit anti-RBC serum were tested for the ability to reverse cytoadhesion of CD7A+I+ and CE11A+I- IRBCs to BBECs. Isotype control mAb HL298, and preimmune rabbit sera were included in the assays as negative controls. Error bars indicate SEMs for two experiments (A) and three experiments (B and C).

 
Because rabbit anti-RBC serum and mAb 4D9.1G1 were both capable of inhibiting cytoadhesion, we wished to determine whether either could disrupt established IRBC-endothelial cell interactions. To ask this question, each Ab source was added to the cytoadhesion assay after the IRBCs had been allowed to adhere. Under these conditions, anti-RBC Abs failed to disrupt the IRBC-BBEC interaction. In contrast, the addition of mAb 4D9.1G1 caused the release of ~90% of adherent CD7A+I+ IRBCs (Fig. 8GoC, p < 0.001), but was without effect on CE11A+I- IRBCs.


   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
Sequestration of B. bovis IRBCs in the microvasculature not only is considered integral to the parasite’s survival in the host (12), but is thought to account for much of the pathology associated with acute disease (11). To study the components mediating sequestration, an in vitro cytoadhesion assay was developed and validated, and several parasite lines and clones that adhere to the endothelial cells were isolated (26). In this study, this model was used to determine the effects of bovine infection sera on cytoadhesion and to begin identification of the IRBC ligands involved in the interaction with endothelial cells.

The immune response to B. bovis infections includes the generation of isolate-specific Igs reactive with the IRBC surface (14, 15, 36). Although anti-IRBC surface Abs are considered important to development of immunity, the mechanistic contributions of these Abs has not been thoroughly addressed. The possibilities include Ab-dependent cellular cytotoxicity killing (5, 6), opsonization (7), and abrogation of sequestration (12). In human infections caused by the related protozoan, P. falciparum, serum reactivity with the IRBC surface is correlated with protection from disease (37, 38). In B. bovis infections, passive transfer of homologous hyperimmune IgG was demonstrated to suppress parasitemias in infected cattle, probably through elimination of IRBCs as well as extraerythrocytic merozoites (4). Unfortunately, the relative contribution of anti-IRBC surface Abs was not assessed, nor was the ability of the serum to reverse sequestration evaluated.

Because of the probable importance of Ig to immunity, the in vitro cytoadhesion assay was used in this study to determine whether Abs generated during infection could disrupt IRBC-endothelial cell bonds. We chose to study clonal lines to ensure uniformity in the IRBC cytoadherence ligands that are expressed. Since neither infection nor hyperimmune sera have yet been raised specifically against the adherent clones, CD7A+I+ and CE11A+I-, infection sera and hyperimmune sera raised against genetically identical clonal lines were used. Infection sera that reacted with the IRBC surface consistently and strongly inhibited adhesion to BBECs. Importantly, these sera could also reverse the binding of IRBCs to BBECs, even under the static conditions of this assay. In addition, the ability of B147 time-course sera to reverse cytoadherence of bound d41A+I- IRBCs was directly correlated with their ability to recognize the IRBC surface in the live-cell IFA. Taken together, these results suggest that Ig responses directed against the IRBC surface are capable of preventing cytoadhesion and even disrupting previously formed bonds between IRBC and endothelial cells. This mechanism may be important in vivo, particularly with the contribution of flow shear, in the clearance of parasites in the immune host.

However, another possible explanation for these results is that serum components other than Ig may affect binding. To test whether the observed inhibition of cytoadhesion was due to serum Igs, B119 IgG and IgG-depleted serum were tested separately for the ability to block binding of IRBCs to BBECs. Significantly, purified IgG effectively inhibited CE11A+I- IRBC adhesion to BBEC. The IgG-depleted serum also contributed to this inhibition, either through IgM, IgA, residual IgG, or other serum factors, as demonstrated by the recovery of full inhibition with the use of IgG-repleted serum. These experiments demonstrate that it is possible for IgG generated during infection to disrupt cytoadhesion, but do not rule out the possibility that components other than Ig may interfere with IRBC-BBEC adhesion. These activities may reflect in vivo mechanisms of immune defense against B. bovis, but confirmation of this role in immunity requires further experimentation.

Previous work demonstrated that export of parasite proteins to the IRBC surface was necessary for adhesion to endothelial cells (26). We therefore sought to identify the exported components. Since B119 infection serum reacted with the IRBC surface and inhibited adhesion as whole serum, as IgG-depleted serum, or as purified IgG, it was used to determine which parasite-synthesized IRBC surface Ags were recognized by each fraction. In surface-specific immunoprecipitations, IgG and IgM from B119 infection serum captured a variable, size polymorphic, high m.w. doublet from both CD7A+I+ and CE11A+I- IRBCs. This doublet, the VESA1 Ag, is a known participant of antigenic variation (3, 17). Accordingly, the size and immunoreactivity of the precipitated Ags varied between these two clones.

Serum that reversed cytoadhesion also precipitated the VESA1 Ag, opening the possibility that expression of this Ag was linked to in vitro cytoadherence. We hypothesized that, if antigenic variation and cytoadhesion were not linked genetically and structurally, then selection for a particular phenotype with regard to one trait should not affect the phenotype of the second trait. However, when time-course sera raised in a chronically infected animal were used to characterize IRBC Ag phenotype following selection for adhesion, the pattern of reactivity was dramatically different between the d41A-I- parent population and the adhesive d41A+I- progeny population (Fig. 4Go). The unique bimodal reactivity with the surface of d41A+I- is unusual. This pattern could reflect successive primary Ab responses to two different combinations of surface epitopes that are expressed on d41A+I- IRBCs, or may be indicative of a classic primary and secondary Ab response to the d41A+I- IRBC surface. Regardless of the explanation, this experiment clearly demonstrates that a parasite population with unique IRBC surface epitopes was isolated by in vitro selection for endothelial cell binding. At least some, and perhaps all changes in IRBC surface reactivity are attributable to changes in the VESA1 Ag. This is clearly demonstrated by the loss of the 4D9.1G1 epitope in the C9.1A-I+ population after selection for the adhesive phenotype. Conversely, selection of MO7A+I- parasites for 4D9.1G1 reactivity resulted in a loss of endothelial cell-binding capabilities. Overall structural changes in the VESA1 Ag expressed by cytoadhesive progeny populations were indicated by corresponding size polymorphisms of the doublet.

More direct support for the hypothesis that VESA1 expression on the IRBC surface is associated with adhesion of IRBCs to endothelial cells was provided by the observation that mAb 4D9.1G1 strongly inhibited and reversed binding of CD7A+I+ IRBCs to BBECs. In contrast, polyclonal anti-RBC membrane Ab could partially inhibit cytoadhesion, probably through steric hindrance, but could not reverse adhesion. Since the 4D9.1G1 epitope is also expressed on erythrocytes infected with the nonadhesive C9.1A-I+ parasite clone, it is unlikely that the mAb epitope per se defines the endothelial cell binding domain. However, this mAb likely binds close to this domain, and with sufficient affinity to effect release of bound IRBCs. Significantly, VESA1 was localized specifically to the tips of the IRBC knob structures, the same site through which cytoadhesion occurs (26). Unambiguous confirmation that VESA1 acts as a cytoadherence ligand for B. bovis IRBCs will require direct evidence that this protein can bind endothelial cells directly, or inhibit binding of IRBCs to endothelial cells when added exogenously. Although the data do not rule out the potential involvement of other IRBC surface components in cytoadhesion, the current evidence is consistent with VESA1 involvement and encourages further exploration of this question.

If the VESA1 Ag serves as cytoadherence ligand for B. bovis IRBCs, then the parasite’s ability to antigenically vary this polypeptide could prove useful not only as a means of evading the immune response, but potentially could enhance the diversity of endothelial cell molecules that could be recognized. When testing the cytoadherence characteristics of primary clones during the cloning of CD7A+I+ and CE11A+I-, different binding phenotypes were observed, suggesting the recognition of more than one endothelial cell ligand by B. bovis-IRBCs (our unpublished observations). Further, size variations in VESA1 were observed among cytoadhesive mAb 4D9.1G1-reactive primary clones possessing different binding phenotypes (our unpublished observations). These observations parallel those made on the related hemoprotozoan, P. falciparum (20, 21, 22), which also sequesters in the host microvasculature (10, 39) through recognition of several endothelial cell receptors (24, 25, 40, 41, 42, 43). The antigenically variant cytoadherence ligand of P. falciparum, PfEMP1, has been observed to contain several conserved DBL domains related to the Duffy-binding adhesive protein (22). The DBL1 domain and associated cysteine-rich interdomain region (CIDR) have been shown to possess CD36 binding activity (23, 44, 45, 46). In addition, a single PfEMP1 isoform could bind to more than one ligand (24, 25). Similarly, thrombospondin, laminin (47), and heparin (48) have been reported to bind B. bovis-IRBCs. However, direct evidence that these compounds are involved in the binding of IRBCs to endothelial cells has not yet been reported. Cloning of the B. bovis cytoadherence ligand, identification of the binding domains, and identification of the endothelial cell receptor(s) will allow a direct comparison of the strategies used by these two protozoans.

Identification of a probable link between cytoadherence and antigenic variation confirms that B. bovis possesses complex mechanisms enabling evasion of host immune defenses. Despite this, the immune host controls the infection (2), suggesting that the genetic potential of the parasite to evade the host immune response does not exceed that of the host to respond to these evasion techniques. Characterization of the receptor-ligand interactions mediating B. bovis sequestration and the immune mechanisms countering this behavior, as well as clarification of all the roles this behavior may play in evading the host immune response, will provide the more complete understanding of the host-parasite relationship needed to better design vaccines and other therapeutic strategies for babesiosis and related diseases.


   Acknowledgments
 
We thank Suzanne Stroup, Ryan Satcher, Paula Patterson, and Jennifer Long for excellent technical assistance.


   Footnotes
 
1 This work was supported by U. S. Department of Agriculture Grants 95-37204-2140 and 97-35204-4768, and by American Heart Association (Florida affiliate) Grant 9810077FL. Back

2 Address correspondence and reprint requests to Dr. David R. Allred, Department of Pathobiology, Box 110880, University of Florida, Gainesville, FL 32611-0880. E-mail address: Back

3 Abbreviations used in this paper: IRBC, infected erythrocyte; BBEC, bovine brain capillary endothelial cells; VESA-1, variant erythrocyte surface Ag 1; NGS, normal goat serum; IFA, immunofluorescence assay. Back

Received for publication September 3, 1999. Accepted for publication December 9, 1999.


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 Results
 Discussion
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