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(Journal of Leukocyte Biology. 2001;70:931-940.)
© 2001 by Society for Leukocyte Biology

Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice to Trypanosoma congolense infection

Karen M. Haas*, Katherine A. Taylor§, Niall D. MacHugh§, John M. Kreeger{ddagger} and D. Mark Estes*,{ddagger}

Departments of
* Molecular Microbiology and Immunology and
{ddagger} Veterinary Pathobiology, University of Missouri, Columbia; and
§ The International Livestock Research Institute, Nairobi, Kenya

Correspondence: D. Mark Estes, Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211. E-mail: EstesD{at}missouri.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.

Key Words: CD40L • trypansome • humoral immunity • VSG • KLH • PBS-T


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypanosomes, such as Trypanosoma congolense, T. brucei, and T. vivax, are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. It is interesting that some breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Cattle breeds belonging to the Bos taurus subspecies, for example, the N’Dama, are able to control parasitemia (trypanotolerant), and Bos indicus cattle, such as Zebu breeds (Boran), succumb to infection [1 ]. Although the basis of trypanotolerance is not yet completely understood, studies have suggested that the immune response elicited against the parasite is involved [1 2 3 ].

Because of its extracellular location, the humoral branch of the immune system dominates the response against T. congolense. Polyclonal B-cell activation is a hallmark of infection, as is elevated immunoglobulin (Ig)M, in bovine and murine trypanosomosis [4 5 6 7 ]. Consistent with this idea, studies performed in BALB/c, nude, and severe combined immunodeficiency (SCID) mice suggest that athymic mice (lacking T cells) can respond to the variable surface glycoprotein (VSG) and under certain conditions, control infection [8 ]. SCID mice lacking B cells and T cells show a relatively high degree of susceptibility versus normal or athymic mice [8 ]. Despite the production of trypanosome-specific antibodies, a large fraction of the IgM that is produced in response to infection is polyspecific and/or autoreactive [4 , 10 11 12 ]. One of the major epitopes to which antibodies are formed during trypanosome infection is the VSG. The importance of VSG-specific antibodies in parasite clearance and protection against infection are supported by a variety of independent studies [13 14 15 16 17 18 ]. Differences have been found to exist between N’Dama and Boran cattle regarding the serum antibody titers and the frequency of cells that secrete antibody reactive against VSG and other trypanosome antigens [2 , 12 , 19 , 20 ]. Although trypanosusceptible Boran animals have been shown to produce more IgM specific for buried and linear epitopes of VSG, the cysteine protease, congopain, and the heat shock protein, (hsp)70, as well as nontrypanosome protein, ovalbumin than do N’Dama cattle, N’Dama cattle are found to produce more IgG1 [2 , 3 , 12 , 21 , 22 ]. This suggests that Boran animals infected with T. congolense are not as effective as trypanoresistant N’Dama cattle at isotype switching to IgG1. This finding may be potentially attributed to the higher level of interleukin (IL)-4 produced by infected N’Dama animals compared with that produced by Boran animals [23 ]. Thus, IL-4, which promotes B-cell switching to IgG1 in cattle [24 ], may play a protective role in the immune response to T. congolense through its ability to stimulate the production of higher specificity antigen-reactive IgG1 by B cells in the presence of CD40L stimulation.

It has been shown previously that in response to T. congolense infection, the percentage of CD5+ B cells in the spleen and blood of cattle becomes increased dramatically [25 ]. The expansion of the CD5+ B-cell subset correlates with the increase in low affinity (often nonspecific), self-reactive serum IgM antibodies, and increased IL-10 transcription that is observed in N’Dama and Boran cattle [26 , 27 ]. IL-10, regardless of what cell population produces it, may play a role in causing the broad immunosuppression that is seen during trypanosome infection. A recent study has demonstrated that compared with preinfection levels of peripheral blood mononuclear cells (PBMCs), CD40L mRNA expression becomes decreased during T. congolense infection of trypanosusceptible and trypanoresistant breeds [23 ]. This broad degree of immunosuppression and the potential lack of CD40L expression as a consequence of the general suppression of T-cell responsiveness during T. congolense infection potentially hinder T-cell-dependent isotype switching by B cells.

We sought to investigate the effect of CD40 stimulation on the bovine immune response generated against T. congolense. Given the difficulty of conducting this type of experiment in cattle, a model of T. congolense infection in SCID-bovine chimeric (SCID-bo) mice was used. The data presented herein characterize the immune response generated during T. congolense infection in the SCID-bo mouse and suggest CD40 stimulation provides protection during trypanosome infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of SCID-bo mice
As described previously, SCID-bo mice were constructed using C.B-17 scid/scid/bg/bg mice reconstituted with bovine hematopoietic cells [28 , 29 ]. All bovine tissues obtained from cross-bred commercial beef cattle (Bos taurus x Bos indicusl; lymph node, liver, and thymus) from second trimester fetuses were surgically implanted into the abdominal cavity of female scid/bg homozygous mice. (Surgeries were performed by Bionova L.L.C., Columbia, MO; mice were obtained from Harlan-Sprague Dawley, Indianapolis, IN.) Prior to surgery, mice received trimethoprim-sulfa suspension (40 mg/5 ml suspension added 1 ml per 32 ml drinking water) and on alternate days, up to 14 days post-operatively. Eight weeks post-implantation, SCID-bo PBMCs isolated over Accupaque gradients (Accurate Chemical, Westbury, NY) were analyzed for the presence of bovine CD45+ cells via flow cytometry [CACTB51A, Washington State University mAb Center, Pullman, WA; rat anti-mouse IgG2a-fluorescein isothiocyanate (FITC), Pharmingen, San Diego, CA]. Prior to staining, nonspecific binding of antibodies to Fc receptors was blocked by preincubation of cells with a blocking antibody reactive against Fc{gamma}II/IIIR (CD16/CD32; Fc Block, Pharmingen). Mice with percentages of bovine cells at 2% or greater than negative isotype-control staining were considered reconstituted and used for experimental purposes.

T. congolense preparation
A frozen African isolate stock of T. congolense (IL3000) was generously supplied by Dr. John Donelson (University of Iowa, Iowa City, IA). The stabilate was injected intraperitoneally (i.p.) into SCID mice to propagate parasites. Upon the development of high-level parasitemia, blood was collected to harvest parasites. Mice were exsanguinated from the retrobulbar sinus under anaesthesia (Metophane, Schering-Plough, Union, NJ). Blood samples containing parasites were diluted 1:1 with phosphate saline glucose (PSG) freezing solution containing 20% glycerol (5.4 g/L Na2HPO4, 0.24 g/L NaH2PO4, 1.7 g/L NaCl, 10 g/L glucose) and were frozen and stored in liquid nitrogen.

Parasite infection of SCID-bo mice
To determine the optimal dose required for T. congolense infection of SCID-bo mice, they were injected i.p. with 5, 50, or 200 parasites in 100 µl phosphate-buffered saline (PBS) and monitored for parasitemia and survival. For all infections that followed, mice were infected with ~80 parasites (diluted in 100 µl sterile PBS) via i.p. administration. Of note, the viability of parasites used in SCID-bo infections was assessed prior to each experiment by microscopy. SCID-bo mice were injected with 50–75 µg protein G-purified (Pierce, Rockford, IL) agonistic mouse anti-bovine CD40 [1LA156 (IgG1), ILRI, Nairobi, Kenya] or mouse IgG1 (MOPC 21, Sigma Chemical Co., St. Louis, MO) on days -1, 0, and 1 post-parasite inoculation. Seven days postinfection, mice were treated with 200 µl 1.12 mg/ml Berenil (diaminazene aceturate; Sigma) i.p. This represents a subcurative dose, because parasitemia eventually redevelops in SCID-bo mice. Fourteen days postinfection, mice were reinfected with ~80 parasites and administered the same anti-CD40 or mIgG1 treatment as described above (on days -1, 0, and 1 of second infection). Seven days post second infection, mice were again treated with Berenil. Blood samples were acquired at weekly intervals to monitor parasitemia.

In all experiments, blood samples collected in heparin obtained by retro-orbital bleeding were centrifuged at 900 g for 20 min, and plasma was removed and stored frozen at -20°C. In some cases, the remaining blood fraction was diluted in 400 µl Hanks’ balanced saline solution (HBSS), overlaid on top of 500 µl Accupaque solution, and centrifuged at 900 g for 20 min. Buffy coats were removed and washed to obtain PBMCs, which were further analyzed by flow cytometric analysis or were used for RNA isolation. A small fraction of blood was also used for determining parasitemia, in which case blood was typically diluted 100-fold in PBS, and parasites were counted using a hemocytometer under 40x ocular lense magnification. Mice exhibiting high levels of parasitemia (107–108) were anaesthesized and sacrificed. Spleens were harvested, ground through stainless steel mesh, passed through 70 µm nylon mesh, overlaid on an Accupaque gradient, and centrifuged to obtain a buffy coat. Spleen buffy coats were harvested, washed in HBSS, pelleted, snap-frozen in liquid N2, and stored at -80°C until subjected to RNA extraction.

Flow cytometric analysis
SCID-bo mice were bled under anaesthesia as previously indicated. PBMCs were isolated by diluting blood 1:1 in room temperature-warmed HBSS and overlaying ~400 µl diluted blood upon 500 µl Accupaque in a 1.5 ml microcentrifuge tube. Samples were centrifuged at 900 g 20 min at room temperature. Buffy coats containing PBMCs were harvested and washed. Prior to staining, nonspecific binding of antibodies to Fc receptors was blocked by preincubation of cells with a blocking antibody reactive against Fc{gamma}II/IIIR (CD16/CD32; Fc Block). Cells were stained with the following antibodies in the following order: anti-CD45 (CACTB51A, WSU mAb Center), anti-mouse IgG2a-FITC (Sigma Chemical Co.), mouse anti-bovine CD5 (CC17, generously supplied by Dr. Chris Howard, IAH, Compton, UK), anti-mouse IgG1-PE (Becton-Dickinson, San Jose, CA), biotinylated anti-bovine IgM (BM23, Sigma Chemical Co.), and streptavidin-allophycocyanin (SA-APC; Pharmingen). Bovine PBMCs were used as a positive triple-stain control, and single stains of bovine PBMCs were used to adjust compensation during analysis of triple stains. Negative triple-stain controls of SCID-bo PBMCs were performed using a combination of mouse IgG2a (Sigma Chemical Co.), anti-mouse IgG2a-FITC, mouse IgG1 (Sigma Chemical Co.), anti-mouse IgG1-PE, and SA-APC. The triple-negative staining control was used to set cursors for quadrant plot analysis. Cells were stained and washed in fluorescein-activated cell sorter (FACS) staining buffer containing 1% bovine serum albumin (BSA) and 0.1% sodium azide azide and were mixed in 2% PBS-buffered paraformaldehyde. Cells were analyzed using a FACS Vantage flow cytometer and Cellquest acquisition and analysis programs (Becton-Dickinson). To obtain fetal lymphocyte populations, fetal tissues (spleen, liver, and mesenteric lymph node) were macerated, and cells were isolated over an Accupaque gradient. Isolated cells were washed in PBS and stained using mouse anti-bovine CD5 (CC17, generously supplied by Dr. Chris Howard, IAH), rat anti-mouse IgG1-PE (Becton-Dickinson), and goat anti-bovine IgM-FITC (Kirkegaard and Perry Labs, Gaithersburg, MD).

Reverse transcriptase-polymerase chain reaction (RT-PCR) of SCID-bo tissues
RNA was extracted according to the manufacturer’s instructions (RNeasy RNA isolation kit, Qiagen, Chatsworth, CA), and DNase was treated with RQ1 DNase (Promega, Madison, WI). DNase-treated RNA was phenol:chloroform-extracted, ethanol-precipitated, and quantitated using a UV spectrophotometer. RNA was then used in RT reactions (1 µg/reaction), according to the manufacturer’s instructions (Perkin Elmer, Foster City, CA). PCR was then conducted on cDNA samples using primers specific for bovine glyceraldehyde 3-phosphate dehydrogenase (G3PDH; Genbank accession #AF077815): forward (nt 1–20), 5'-GGAAGCTCACTGGCATGGCC-3'; reverse (nt 274–294), 5'-CCCTGTTGCTGTAGCCAAA-3', and IL-10 (Genbank accession #U00799): forward (nt 81–102), 5'-AGCTGTATCCACTTGCCAACC-3'; reverse (nt 434–454), 5'-TCTCTTGGAGCTCACTGAAG-3' (40 cycles/reaction). Because of the high degree of homology between the murine and bovine housekeeping gene, G3PDH, identification of a restriction-length fragment polymorphism was necessary to differentiate between bovine and murine G3PDH cDNA. An MspI (i.e., HpaII) restriction site was identified in the murine G3PDH sequence that was absent in the corresponding bovine G3PDH cDNA. Digestion of primed G3PDH RT-PCR cDNA product yields a murine cDNA product of 225 bp compared with an undigested bovine G3PDH fragment of 294 bp. MspI-restriction digestion of cDNA generated from G3PDH-primed RT-PCR reactions was performed for 1.5–2 h at 37°C. IL-10 RT-PCR samples as well as digested G3PDH RT-PCR samples were analyzed by resolution on an ethidium bromide-stained 2% Tris-acetate EDTA (TAE) agarose gel, followed by Southern hybridization. Southern hybridization was performed by capillary transfer of nucleic acids to nylon membrane (GeneScreen Plus II, NEN, Boston, MA), followed by UV cross-linking. Membranes were prehybridized in PerfectHybPlus containing heat-denatured herring sperm DNA (100 µg/ml final; Sigma Chemical Co.) for 4 h at 47°C, followed by addition of 32P-labeled probes overnight, generated using cloned bovine fragments of bovine G3PDH (nt 1–294), IL-10 (nt 81–454), and a random-primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IA). Blots were washed three times in 2 x saline sodium citrate (SSC) at room temperature and exposed to film (X-OMAT AR, Eastman Kodak Co., Rochester, NY) in the presence of intensifying screens. Where indicated, densitometric analysis was performed on autoradiographs using Kodak digital imaging/analysis software (Eastman Kodak Co.).

Histology
Spleens fixed in 10% buffered formalin (Fisher Scientific, St. Louis, MO) were embedded in paraffin, and 5 µm sections were cut. A portion of sections were stained with hematoxylin and eosin, and others were individually and immunohistochemically stained. Sections to be used for immunostaining were pretreated with citrate buffer, pH = 6 (DAKO, Carpinteria, CA) in a steamer. Spleen sections were stained to detect CD3 (rabbit anti-human CD3; 1:600, DAKO) and bovine IgM/IgG/IgA (1:200; mouse anti-bovine IgM, IgG, IgA, Serotec, Raleigh, NC) using an avidin-biotin-peroxidase system (LSAB-2 kit, DAKO) and peroxidase-3,3'-diaminobenzidine (DAB) development.

Dot blot antibody detection
Plasma from SCID-bo mice was diluted (1:25 in PBS) and blotted onto a pre-wetted nitrocellulose membrane using a dot blot manifold to detect total antibody present in plasma. Membranes were then pre-wet in PBS and blocked for 1–2 h at room temperature in Superblock (Pierce, Rockford, IL). Following brief washes in PBS + 1% Tween (PBS-T), membranes were incubated with goat anti-bovine IgM-horseradish peroxidase (HRP; diluted 1:1000 in PBS-T; Kirkegaard and Perry Labs) or goat anti-bovine IgG-HRP (diluted 1:2000 in PBS-T; Jackson Immunoresearch, West Grove, PA) for 30 min at room temperature. Following 45 min of vigorous washing in PBS-T, blots were developed using enhanced chemiluminescence (ECL+kit, Amersham, Piscataway, NJ), and signals were detected using Kodak X-OMAT AR film.

Antigen-specific antibody was detected in SCID-bo plasma in a similar manner. Antigen [100 ng IL3000 VSG, ovalbumin (Sigma Chemical Co.), or keyhole limpet hemocyanin (KLH; Pierce)] was blotted onto pre-wetted nitrocellose. Membranes were dried, blocked, and incubated with diluted SCID-bo plasma (1:25–1:50) for 1 h at room temperature. Following 45 min of vigorous washing, blots were treated as above (i.e., incubation with anti-bovine IgM- or IgG-HRP followed by washing and addition of substrate).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of parasite dose
To determine the optimal number of parasites to use for infection experiments, SCID-bo mice were infected with varying numbers of the bloodstream form of T. congolense. Chimeric mice were evaluated prior to infection by flow cytometry in order to determine relative reconstitution on the basis of the percentage of peripheral blood mononuclear cells expressing bovine CD45 (Table 1 ). Mice were assigned randomly to the study groups. Studies of T. congolense infection conducted in susceptible BALB/c mice have shown that infection with 103 parasites i.p. results in parasitemia in less than 7 days and a survival time of approximately 8 days [30 ]. In an initial experiment, 800, 8000, or 80,000 parasites were injected via an i.p. route into SCID-bo mice. Given the rapid multiplication of the parasite and its efficient entry into the blood, mice became parasitemic and moribund within less than 1 week of infection (unpublished results). In the experiment that followed, SCID-bo mice (two per group) were infected with 5, 50, or 200 parasites i.p. and monitored for the development of parasitemia (i.e., the presence of parasites in the blood). As shown in Figure 1 , mice infected with 200 parasites developed parasitemia within 8 days of the initial infection. Mice given 50 parasites i.p., however, survived 9–11 days postinfection. Finally, mice infected with approximately five parasites did not develop parasitemia within the first 2 weeks of infection and differed greatly in the time frame at which parasitemia developed. Because of the inability to ensure that equal numbers of parasites were injected into SCID-bo mice when such low parasite numbers were used (approximately five parasites), an intermediate dose (80 parasites) between 50 and 200 parasites was chosen as an infectious dose for further experiments, because it would likely produce consistent development of infection in SCID-bo mice but would allow mice to survive beyond 1 week following infection.


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  		Table 1. Representative Assessment of the Percentage of boCD45+ Cells by Flow Cytometry in SCID-bo Mice for Various Treatment Groups from Fetal Tissue Donors 1 and 2 at 8 Weeks Post-Surgery

 


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  		Figure 1. Infection of SCID-bo mice with T. congolense. SCID-bo chimeric mice (two per group) were infected with ~200, 50, or 5 parasites i.p. and monitored for the development of parasitemia over time. Parasitemia was determined by taking a small blood sample via retro-orbital bleed under anaesthetic and counting parasites under 400x magnification by phase contrast microscopy.

 
Survival and the development of parasitemia in SCID-bo mice
In a separate series of experiments, SCID-bo mice were infected i.p. with ~80 T. congolense parasites, and on days -1, 0, and 1 postinfection, 50 µg anti-CD40 (ILA-156), an agonistic antibody, or control mouse IgG1 was administered i.p. One week following the start of infection, mice were treated i.p. with Berenil, a drug that exhibits trypanocidal activity. Mice were treated with a subcurative dose of Berenil at 7 days postinfection to prolong survival such that the immune response generated against the parasite infection would have sufficient time to develop. Seven days post-Berenil treatment, mice were reinfected with 80 parasites and administered antibody as performed during the primary infection. Seven days following this, mice were again Berenil-treated and monitored for the development of parasitemia and survival. As shown in Figure 2 , 27 days following the start of the experiment in which SCID-bo mice from donor 1 was used (14 days following the second infection), mice began to die as a result of infection. It is interesting that by 30 days post-primary infection, 5/6 anti-CD40-treated mice had survived, and only 2/6 mice receiving the control isotype (mIgG1) remained, suggesting that anti-CD40 treatment afforded mice some degree of protection. At this timepoint (30 days), however, remaining SCID-bo mice in both treatment groups began to succumb to parasitemia and were thus sacrificed for analysis. In a second experiment (fetal tissues from SCID-bo mouse donor 2), a similar infection and treatment schedule was used in which 75 µg anti-CD40 or mouse IgG1 control antibody was administered to mice i.p. Mice were monitored for survival or parasitemia levels (unpublished results) greater than 1 x 108 parasites/ml, at which time mice were sacrificed. A survival plot for this experiment (SCID-bo mice with fetal tissue implants from donor 2) is shown in Figure 2 . Similar to the results obtained for the infected SCID-bo mice constructed using donor 1, mice constructed from donor 2 displayed enhanced survival (or maintained lower levels of parasitemia) when anti-CD40 treatment was administered. The difference in survival kinetics observed between the first and second experiment may be attributable to such factors as donor variability, reconstitution efficiency, and/or parasite viability. Of note, in a third experiment, the effect of withholding Berenil treatment was demonstrated to result in the earlier development of parasitemia. In this particular experiment, a difference in survival among antibody treatments was not observed (unpublished results). Thus, in the absence of the trypanocidal drug, infection ensues at a more rapid pace, potentially obscuring any protective effect antibody treatment may have in the SCID-bo mouse. However, in combination with Berenil treatment that effectively reduces parasitemia, CD40 stimulation elicits a protective effect during trypanosome infection in SCID-bo mice.



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  		Figure 2. Survival curve of SCID-bo mice infected with ~80 T. congolense parasites i.p. Six SCID-bo mice reconstituted with tissues from fetal donor 1 or donor 2 were treated with anti-CD40 (50 µg in donor 1, 75 µg in donor 2) or the isotype control mouse IgG1(mIgG1) i.p. on days -1, 0, and 1 postinfection. Seven days postinfection, mice were treated with Berenil to reduce parasite load. Seven days following treatment (14 days postinfection), mice were again infected with ~80 parasites i.p. Antibody treatment was administered at days -1, 0, and 1 post second infection (i.e., days 13, 14, and 15 post initial infection). Seven days following the second infection (i.e., day 21), mice were again treated with Berenil to reduce parasite load. Mice were monitored for development of parasitemia and survival. At day 30 (donor 1) or day 28 (donor 2), remaining mice were sacrificed for analysis. In the experiment shown in the donor 1 graph, six mice were used per treatment group, and in the experiment shown in the donor 2 graph, the anti-CD40 group consisted of seven mice, and the mIgG1 isotype control group consisted of eight mice.

 
Lymphocyte populations of bovine fetal tissues
Based on the observation that the CD5+ B-cell population becomes expanded during T. congolense infection in cattle [25 ], we were interested in determining the effect of parasite infection on the B-cell populations of SCID-bo mice. Although the relative percentage of bovine IgM+ cells in the peripheral blood of SCID-bo mice has been demonstrated previously [29 ], the percentage of B cells expressing CD5 in these mice had not yet been investigated. In addition, the percentage of B cells expressing CD5 in fetal tissues used for implantation was not known. Thus, tissues from second trimester bovine fetuses were analyzed for the presence of CD5+IgM+ cells. Flow cytometric analysis of B lymphocyte populations within second trimester fetal tissues indicates that small percentages of B-cell populations expressing IgM are present at this stage of development as shown in Figure 3 . Fetal spleen (not used for implantation) is populated by few IgM-expressing cells (~2%). Lymph node (mesenteric) and liver (tissues used for SCID-bo implantation), however, are populated by larger percentages of B cells (~10%). It is interesting that the majority of IgM+ cells found in the lymph node and liver expresses CD5. T cells are found in the CD5hiIgM- population, constituting approximately 20% of the cell population found in the fetal lymph node and liver.



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  		Figure 3. B-cell populations of second trimester fetal tissues. Lymphocyte populations from spleen, lymph node, and liver tissues from a second trimester bovine fetus were analyzed for CD5 and IgM expression by flow cytometry. Fetal tissues were macerated, and a single-cell suspension was isolated using an Accupaque gradient. Cells were then stained and analyzed by flow cytometry as described in Materials and Methods.

 
B-cell populations of SCID-bo mice in response to T. congolense infection
In addition to monitoring parasitemia and survival of trypanosome-infected SCID-bo mice, changes in bovine B-cell populations in the peripheral blood of SCID-bo mice during infection were analyzed by flow cytometry. PBMCs were isolated at various timepoints and triple-stained using antibodies against bovine CD45, CD5, and IgM. Nonspecific binding of antibodies to Fc receptors was blocked by preincubation of cells with a blocking antibody reactive against Fc{gamma}II/IIIR (CD16/CD32). The level of CD5 and IgM expression of bovine cells was conducted by limiting the analysis of CD5 and IgM expression to the bovine CD45+-gated cell population. The triple-negative staining control was used to set cursors for quadrant plot analysis. The percentage of total bovine (boCD45+) IgM+ cells and percentage of bovine IgM+CD5+ cells are shown for two experiments in Figure 4 A . As indicated by the graphs, the majority of bovine IgM+ B cells in the SCID-bo mouse expresses CD5. This is similar to what we observed in this study for the bovine fetal liver and lymph node IgM+ populations (Fig. 3) as well as the neonatal B-cell populations of mouse and human [31 , 32 ]. No significant differences were observed in the total percentage of bovine B cells, or the PBMC populations that were also analyzed in a similar manner at 14 days post initial infection for tissue donor 1- and tissue donor 2-recipient SCID-bo mice. As demonstrated in Figure 4A , similar to the observation made for pre-infection populations, nearly all (>90%) IgM+ B cells coexpressed CD5. However, differences were observed in the total percentage of IgM+CD5+ cells found in peripheral blood among treatment groups. As indicated by the graph in Figure 4A , anti-CD40-treated mice had a higher mean percentage of total IgM+ bovine cells and IgM+CD5+ cells in peripheral blood than the mIgG1 control group 14 days postinfection. Because of the number of mice used in the first experiment shown (anti-CD40-treated, n=4; isotype control group, n=5), this difference is not statistically significant using a Student’s t-test with a 95% confidence interval. However, the difference among treatment groups in the mean total percentage of IgM+ cells as well as the mean CD5+ IgM+ cell percentages 14 days postinfection in the experiment shown for SCID-bo mice from donor 2 were found to be statistically significant (mean percentage IgM+ cells, P<0.01; mean percentage CD5+IgM+ cells, P<0.05; Student’s t-test). Increases in the percentage of bovine B cells in the peripheral blood of infected SCID-bo mice treatment were also observed in a third experiment in which SCID-bo PBMCs were analyzed at 22 days postinfection (unpublished results). The percentage of CD5+IgM+ cells in the SCID-bo mouse among treatment groups prior to infection was consistently around 40% the total CD45+ cells in the circulating peripheral blood pool. The percentage of B cells in individual mice was increased, but variable at day 22 postinfection in the one experiment in which this parameter was evaluated (36+18% vs. 24+12%) (donor 1).



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  		Figure 4. Peripheral blood B-cell populations of SCID-bo mice on day 0 and days 14 and 15 postinfection. PBMCs were isolated from SCID-bo mice and analyzed for the expression of bovine CD45, CD5, and IgM. The percentage of bovine CD45+-gated cells expressing IgM and the percentage coexpressing IgM and CD5 were determined for individual mice and were averaged to determine the mean percentage of IgM+ cells (total B cells) as well as the mean total percentage of IgM+CD5+ cells (CD5+ B cells). (A) Analysis of B-cell populations from trypanosome-infected SCID-bo mice at day 0 (pre-infection) and day 14 following anti-CD40 treatment or mouse IgG1 control antibody. The experimental data shown for SCID-bo mice constructed from fetal donor 1 consist of five mice receiving 50 µg mIgG1 isotype control antibody and four mice receiving 50 µg anti-CD40 treatment on days -1, 0, and 1 of infection and Berenil treatment seven days postinfection. The experimental data shown for SCID-bo mice constructed from fetal donor 2 consist of six mice receiving 75 µg mIgG1 isotype control antibody and six mice receiving 75 µg anti-CD40 treatment on days -1, 0, and 1 of infection and Berenil treatment 7 days postinfection. (B) Flow cytometric analysis of B-cell population PBMCs from noninfected SCID-bo mice (n=3) prior to anti-CD40 administration and 15 days following anti-CD40 treatment (75 µg administered i.p. on days 1, 2, and 3). (A and B) Error bars represent the standard deviation of calculated means. Statistical analysis was performed using a Student’s t-test.

 
However, analysis of PBMCs at an earlier timepoint (6 days postinfection) did not reveal any differences in CD5+ B cells or total IgM+ B cells between anti-CD40 and isotype control groups (unpublished results). Thus, CD40 stimulation was observed to expand the bovine B-cell populations of infected SCID-bo mice 14 days postinfection. Importantly, anti-CD40 stimulation alone (in the absence of infection) was not observed to expand B-cell populations in SCID-bo mice (Fig. 4B) .

Immunohistochemical analysis of T. congolense-infected SCID-bo mice
To determine the effect of anti-CD40 treatment on splenic architecture and bovine lymphocyte populations within the spleen of infected SCID-bo mice, formalin-fixed spleen sections were embedded in paraffin, sectioned, and visually analyzed by hematoxylin and eosin or immunohistochemical staining. Hematoxylin/eosin stains indicated that lymphoid cells were indeed localized in the white pulp areas of SCID-bo spleens of mouse IgG1 control and anti-CD40-treated mice (unpublished results). To determine the contribution of bovine T cells to the splenic white pulp, spleen sections were stained with an antibody specific for CD3. Staining results are shown for the isotype-control-treated group in Figure 5 as indicated. CD3+ cells were detected in defined areas surrounding central arterioles throughout the spleen. T cells were mainly localized around arterioles in T-cell zones, although a few T cells were found outside these clusters. Subjective analysis of stained sections indicated no discernible differences in T-cell numbers. Finally, an antibody specific for bovine IgM, IgG, and IgA was used to detect the presence of B cells within spleens of infected SCID-bo mice. The staining results are shown in Figure 5 ({alpha}Ig). Fewer numbers of B cells, relative to CD3+ T cells, were detected in spleens. However, B cells appeared to be more abundant in anti-CD40-treated mice compared with the mIgG1 control group. This finding correlates well with the increase in B cells observed in the peripheral blood of anti-CD40-treated mice, as detected by flow cytometry. The paucity of B cells in the spleens of SCID-bo mice, relative to T cells, has been demonstrated previously in a SCID-hu model of lymphocyte reconstitution. In this particular study, human B cells (expressing CD5) were lacking in spleen but were found localized in the thymus [33 ]. An attempt was made to analyze remnants of thymus but was unsuccessful as a result of the inability to consistently identify thymic tissue in SCID-bo mice. Alternative locations of B cells, such as the peritoneal cavity, remain to be investigated.



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  		Figure 5. Detection of T and B cells in spleens of T. congolense-infected SCID-bo mice from fetal tissue donor 2. Sections of paraffin-embedded spleens from control (mIgG1) or anti-CD40-treated SCID-bo mice were stained with a species cross-reactive rabbit anti-human CD3 polyclonal antibody (DAKO) or a mouse monoclonal antibody specific for bovine IgM, IgG, and IgA (Serotec) and developed using an avidin-biotin-peroxidase system and DAB as a substrate. Representative stains are shown at 40x original magnification.

 
IL-10 mRNA production by SCID-bo mice
Because IL-10 is observed to be produced at high levels during T. congolense infection, IL-10 mRNA levels were analyzed for PBMCs and spleen at the peak of infection (i.e., when mice became moribund or severely parasitemic). RT-PCR Southern blotting was used to detect the presence of IL-10 mRNA. G3PDH RT-PCR was used to control for variation in the levels of bovine mRNA present in spleen and blood samples. As described in Materials and Methods, restriction-length fragment polymorphism analysis of bovine and murine G3PDH indicates that MspI is capable of cleaving the murine but not bovine G3PDH cDNA product. Thus, to differentiate between bovine and murine G3PDH RT-PCR products, cDNA was subjected to MspI restriction digestion prior to resolution by agarose gel electrophoresis and detection by Southern blotting. IL-10/G3PDH Southern blots performed on individual SCID-bo spleen samples from donors 1 and 2 are displayed in Figure 6 A . It is evident that a great deal of variation exists between signals for IL-10 and G3PDH among individual mice. However, an appreciable signal is detected for IL-10 in both treatment groups. IL-10 signal was detected for pooled mRNA from both treatment groups, although PBMCs from mouse isotype control group displayed more intense IL-10 signals. The relative difference in signals among treatment groups was determined by densitometric analysis, whereby the ratio of IL-10 to G3PDH signals was determined. The difference in IL-10:G3PDH ratios among treatment groups is shown in Figure 6B . As shown for experiment 1, no difference was detected between anti-CD40 and mIgG1 antibody treatments. In experiment 2, anti-CD40-treated mice had a slightly lower ratio of IL-10 to G3PDH than the mIgG1-treated group. Finally, the IL-10:G3PDH ratio was found to be lower in the anti-CD40-pooled PBMCs compared with the isotype control group-pooled PBMCs. In addition to IL-10 mRNA analysis, an attempt was made to detect IL-4 mRNA, however no signals were detected by RT-PCR Southern blotting.



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  		Figure 6. Detection of IL-10 mRNA in T. congolense-infected SCID-bo mice. (A) RNA was extracted from the spleens or PBMCs of infected SCID-bo mice at the time of sacrifice and analyzed by RT-PCR/Southern blotting. Following DNase treatment, extraction, and precipitation, 1 µg mRNA was used in a reverse transcription reaction. Bovine-specific IL-10 primers and G3PDH primers were then used to prime and amplify reverse-transcribed cDNA. G3PDH cDNA was subjected to MspI digestion to cleave mouse but not bovine G3PDH cDNA products. PCR products were then resolved on a 2% agarose gel and blotted onto nylon membrane. Membranes were probed with 32[P]-labeled bovine IL-10 and G3PDH DNA probes and developed by autoradiography. Results are shown for splenic mRNA isolated from individual SCID-bo mice reconstituted from fetal donor 1 and donor 2. The RT-PCR Southern blot for pooled PBMC mRNA for SCID-bo mice reconstituted from donor 2 is also shown. (B) Quantitation of the ratio of IL-10 signal to G3PDH signal for experiments 1 and 2. Densitometric analysis was performed on the autoradiographs for SCID-bo spleen and PBMC samples of experiments 1 and 2 shown in (A). Using a Kodak digital analysis system, the net intensities of IL-10 and G3PDH bands were determined. The ratio of the IL-10 to G3PDH signal was determined for each individual mouse spleen sample, averaged for each treatment group in experiments 1 and 2, and graphed. The ratio of the IL-10-to-G3PDH signal was also determined for the pooled PBMCs and plotted. Error bars represent the standard deviation of the mean.

 
Detection of antibody in plasma of T. congolense-infected mice
Given the importance of antibody in the response against T. congolense, we sought to analyze antibody responses against VSG, an immunodominant antigen, in the SCID-bo mouse. Thus, an attempt was made to detect VSG-specific antibody in the plasma of SCID-bo mice using a dot blot developed by enhanced chemiluminescence. However, plasma collected from SCID-bo mice at the time of sacrifice did not yield positive results for VSG-specific antibody. As a result of the high level of parasitemia and, therefore, high antigen load, we were interested in examining the total level of bovine antibody in the plasma of SCID-bo mice. As shown by the dot blot in Figure 7 A , although IgM and IgG antibodies are detected in the serum of SCID-bo mice 14 days postinfection, at the time the mice were killed when parasitemia levels were at their highest, antibody is largely undetectable. Thus, SCID-bo plasma was analyzed at 14 days postinfection for the presence of VSG-specific antibody. Additionally, the presence of polyclonal antibody reactive against ovalbumin or KLH in SCID-bo plasma was examined. As demonstrated in Figure 7B , IgM reactive against VSG was detected in all SCID-bo mice, infected and noninfected. Importantly, a previous study has demonstrated that trypanosome VSG-reactive antibodies are found in the sera of uninfected hosts of multiple species [34 ]. It is interesting that at this level of sensitivity, ovalbumin- and KLH-reactive IgM was also present in all SCID-bo mice. Thus, a portion of the IgM produced by bovine B cells in the SCID-bo mice is potentially a result of bystander B-cell activation resulting in a generalized increase in this isotype. In contrast, bovine IgG specific for VSG was detected only in the plasma of infected SCID-bo mice. In addition to VSG-specific IgG, IgG reactive against KLH and ovalbumin was also detected only in infected SCID-bo mice. Thus, similar to that observed for T. congolense-infected cattle and mice [4 , 11 , 12 , 27 ], nonspecific antibody responses are made by SCID-bo mice infected with T. congolense. However, anti-CD40 treatment appeared to result in increased production of IgG, VSG-specific and nonparasite antigen-specific, as detected by dot blot analysis.



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  		Figure 7. Detection of SCID-bo antibody by dot blot analysis. (A) Total antibody present in SCID-bo plasma at 14 days postinfection and at the time of sacrifice. Plasma from SCID-bo mice was pooled and diluted (1:25 in PBS) and 10 µl blotted onto pre-wetted nitrocellulose. Membranes were blocked in Superblock, washed, and incubated with goat anti-bovine IgM-HRP or goat anti-bovine IgG-HRP. Following vigorous washing in PBS-T, blots were developed using ECL + kit, and signals were detected using Kodak X-OMAT AR film. Results are shown for two experiments in which 50 µg (exp. 1)/75 µg (exp. 2) of anti-CD40 (ILA156) or mouse IgG1 was administered to mice as described in Materials and Methods. (B) Detection of VSG and nontrypanosome antigen-specific antibody responses in SCID-bo mice. VSG (100 ng), ovalbumin (OVA), or KLH was blotted onto nitrocellulose. Membranes were blocked (Superblock) and incubated with pooled plasma diluted 1:50 (upper blot) or 1:25 (lower blot). Bound antibody was detected as described in (A). Data are shown for plasma taken from anti-CD40 or mIgG1-treated SCID-bo mice 14 days following parasite infection (7 days following Berenil treatment). Data are also shown for plasma from noninfected SCID-bo mice receiving anti-CD40 treatment (d14). Normal, noninfected C.B-17 serum was used as a negative control. Results are representative of independent experiments that used mice reconstituted with fetal tissues from two individual donors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Trypanosomosis caused by the extracellular parasite, T. congolense, is characterized by expansion of the CD5+ B-cell subset [23 ], elevated production of IgM [4 5 6 7 ], a large fraction of which is polyspecific and/or autoreactive [4 , 10 11 12 ], and increased IL-10 transcription [26 , 30 ]. Additionally, T. congolense infection results in a broad degree of immune suppression [4 , 35 36 37 ]. Recently, Mertens et al. [23 ] demonstrated that cattle infected with T. congolense exhibited decreased levels of CD40 ligand mRNA expression early in infection. The consequence of reduced CD40L expression during trypanosome infection is unknown presently, although it can be speculated that B-cell responses requiring CD40-CD40L interactions such as isotype switching and clonal expansion would be hindered. However, the role of CD40-CD40L interactions during T. congolense infection in cattle had not been investigated previously. Thus, we aimed to address this question using a SCID-bo chimeric mouse model.

To begin to study the B-cell responses that occur in this model during trypanosome infection, we sought to first analyze the B-cell populations (CD5+ or CD5-) present in the SCID-bo mouse. An important finding shown here is that the overwhelming majority of bovine IgM+ B cells in the SCID-bo mouse expresses CD5. Analysis of bovine fetal tissues indicated that a significant portion of the IgM+ B-cell populations in the second trimester bovine fetal liver and lymph node express CD5. This is similar to what has been demonstrated for human fetal liver and lymph node [38 , 39 ]. Fetal spleen tissue, however, was virtually devoid of B cells during the second trimester of gestation. Given that fetal thymus, liver, and lymph node tissues are used to reconstitute the immune repertoire of SCID mice, it is not surprising to discover that the majority of bovine B cells reconstituting the peripheral blood lymphocyte population of the SCID mouse expresses CD5. Thus, this reconstituted B-cell population may resemble that of the neonate. The process and rate of maturation of the immune system within the reconstituted SCID-bo mouse in comparison to that which occurs in the neonatal calf remain to be established.

Initially we had intended to use the SCID-bo model to study the conversion of a bovine CD5- B-cell phenotype to a CD5+ phenotype as a consequence of T-cell-independent type 2 (TI-2)-antigenic B-cell activation that may potentially occur during trypanosome infection [8 , 40 ]. However, given the finding that the majority of SCID-bo B cells expresses CD5 prior to infection, we were unable to explore this process of B-cell differentiation using the SCID-bo model. However, the findings obtained using this model to study trypanosome infection were nonetheless exciting. Although no difference among treatment groups was observed with respect to the percent of IgM+ cells expressing CD5, increases in the total percentage of IgM+ B cells and CD5+IgM+ B cells in the peripheral blood of SCID-bo mice treated with anti-CD40 antibody were consistently observed during T. congolense infection. Importantly, in the absence of infection, anti-CD40 treatment was not observed to increase the percentage of B cells in the peripheral blood of SCID-bo mice. Thus, CD40 stimulation did not decrease the relative percentage of B cells expressing CD5 but actually served to expand the overall percentage of CD5+IgM+ B cells as well as the overall percentage of CD5-IgM+ B cells, given that the percent of IgM+ cells, which expresses CD5, remained relatively consistent. This finding suggests that anti-CD40 treatment, although unable to reverse the development of CD5+ B cells, did indeed stimulate B cells (CD5+ and CD5-) to expand in vivo concomitant with parasite infection. It is interesting that similar to anti-CD40-treated SCID-bo mice, trypanoresistant N’Dama cattle are shown to have a higher proportion of B cells during infection than similarly infected susceptible Boran cattle [2 ].

Consistent with studies of high levels of IL-10 production in murine and bovine trypanosomosis [26 , 30 ], IL-10 mRNA expression in the spleen and PBMCs of infected SCID-bo mice was elevated. Previously, IL-10 has been suggested to play a role in the immunosuppression observed during trypanosomosis [30 , 37 ]. Macrophages have been implicated in the production of IL-10 during trypanosome infection [41 ], and T cells and CD5+ B cells may potentially produce IL-10 as well. However, the cell type(s) responsible for producing IL-10 during trypanosome infection in SCID-bo mice remains to be determined.

Importantly, this study demonstrates that IgG antibody specific for VSG is produced by T. congolense-infected SCID-bo mice. Although IgM antibodies reactive against VSG, as well as ovalbumin and KLH, were present in the plasma of uninfected and infected SCID-bo mice, IgG-reactive antibody was only present in T. congolense-infected animals. The presence of VSG-reactive IgM antibody in SCID-bo mice that have not been exposed to T. congolense is not an unexpected finding, because self-reactive antibodies that recognize VSG have been shown previously to exist in the serum of uninfected hosts [34 ]. CD40 stimulation (via ILA156 administration), as expected, was observed to increase the level of IgG detected in the plasma of infected mice compared with mice receiving the isotype control. As stated earlier, this increase in VSG-specific IgG may have afforded mice some degree of protection during parasite infection. Importantly, VSG-reactive antibody was only detected in the plasma of mice 14 days postinfection (7 days post initial Berenil treatment). During high levels of parasitemia, antibody was difficult to detect in the plasma of SCID-bo mice. This is likely a result of the fact that bovine antibody is rather limiting in reconstituted mice and that under conditions of high parasitemia, antibody is rapidly depleted because of high antigen load and clearance.

The results presented for the experiments involving SCID-bo T. congolense infection have interesting implications regarding the SCID-bo model as well as for T. congolense infection. The data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection. Although the reasons for this observed effect remain to be determined, it can be speculated that by increasing the level of isotype switching to VSG-specific IgG and by increasing the total number of B cells, CD40 stimulation may have enhanced parasite clearance via overall increased IgG antibody production. Indeed, although trypanosusceptible Boran cattle have been shown to produce more IgM specific for buried and linear epitopes of VSG the cysteine protease, congopain, and hsp70, as well as nontrypanosome protein, ovalbumin than do N’Dama cattle, N’Dama cattle are found to produce more IgG1 [2 , 3 , 12 , 21 , 22 ]. This suggests that Boran animals infected with T. congolense are not as effective at isotype switching to IgG1 as trypanoresistant N’Dama cattle. Thus, similar to what is observed in cattle, isotype selection may play an important role in providing protection during T. congolense infection in the SCID-bo mouse. Additional studies aimed at addressing the role of isotype switching, as well as the factors influencing switching during trypanosome infection are required to develop an understanding as to how cattle may achieve protection via antibody during infection. The SCID-bo model provides a useful tool for studying the development of trypanosomosis in cattle. Through the ability to manipulate the immune response of these heterochimeric mice via antibody or cytokine administration, the factors involved in providing protection during T. congolense infection may be more clearly elucidated.


   ACKNOWLEDGEMENTS
 
This work was supported by a University of Missouri-COR grant (#2-05028) provided to K. M. H. and D. M. E. and by a United States Agency for International Development grant provided to K. A. T., K. M. H., and D. M. E. We thank Dr. John Donelson (University of Iowa) for supplying T. congolense (IL3000) as well as Dr. Chris Howard (IAH) for supplying CC17. We also thank Louise Barnett for her assistance with flow cytometric analysis as well as Marilyn Beissenherz for her assistance with immunohistochemistry.

Received April 7, 2001; revised July 23, 2001; accepted August 3, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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K. C. Lambert, E. M. Curran, B. M. Judy, G. N. Milligan, D. B. Lubahn, and D. M. Estes
Estrogen Receptor {alpha} (ER{alpha}) Deficiency in Macrophages Results in Increased Stimulation of CD4+ T Cells while 17{beta}-Estradiol Acts through ER{alpha} to Increase IL-4 and GATA-3 Expression in CD4+ T Cells Independent of Antigen Presentation
J. Immunol., November 1, 2005; 175(9): 5716 - 5723.
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