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Keywords:

  • bovine;
  • Babesia;
  • innate immunity;
  • spleen;
  • cytokine mRNA expression

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Young calves possess a strong innate immunity against Babesia bovis infection that lasts for approximately 6 months after birth and is abrogated with the removal of the spleen. This immunity is characterized as cellular involving a soluble mediator. Nitric oxide has been implicated by virtue of its babesiacidal affects in vitro, but questioned to be as effective in vivo, due to its ability to downregulate type-1 immunity. Spleen cells were obtained from 4-month-old calves and adult steers and processed for monitoring cytokine and inducible nitric oxide synthase (iNOS) mRNA expression during the response to initial B. bovis infection. The data provided evidence of a transient role for nitric oxide in innate immunity, characterized by brief iNOS induction in the spleen of calves that was not detectable in the spleens of adults. The iNOS message followed the early induction of interleukin (IL)-12 and interferon (IFN)-γ message in calves. The induction of IL-12 and IFN-γ message in adults was delayed until IL-10 message was induced. Transformation growth factor-β mRNA expression levels were greater in spleen cells from adults early in infection and then declined, whereas expression levels increased in spleen cells from calves later in the infection process. Together, the data support the concept of ‘first come, first serve’ cytokine influence over cellular activities, the importance of a type-1 response in the control of an initial infection and the need for tight regulation in order to prevent pathology associated with over production of nitric oxide and inflammatory cytokines.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Babesiosis is a tick-borne, haemoparasitic disease, which in cattle is of economic importance throughout the world, especially in tropical and subtropical regions (1). The studies reported here involve Babesia bovis, one of six recognized species that can infect cattle (2).

The immune response directed against infections with intraerythrocytic parasites, such as Plasmodium and Babesia, involves both humoral and cellular mechanisms (3) and is T-cell-dependent (4–9). In addition, an age-related immunity to initial infection with B. bovis in cattle is well established, characterized by strong innate immunity in young calves (10). There is a cellular component to this age-related immunity (11,12) that involves a soluble babesiacidal factor (13).

Mononuclear phagocytes (MP) are engaged as the primary effector cells of innate and primary immune responses and nitric oxide (NO) has been identified as at least one babesiacidal molecule produced by activated MP (14–16). When B. bovis infected erythrocytes grown in culture are exposed to NO, either in a cell-free system (17) or in the presence of activated MP (16), death of the parasites (crisis forms) occurs rapidly within the erythrocytes. In addition, B. bovis merozoites cultured with bovine MP can induce NO production with a babesiacidal effect (18). Because NO is very short lived, it is suspected that the microbicidal effects are not systemic but occur in lymphoid organs such as the spleen where distance between effector cell and target is minimal (19). The microbicidal effects can occur in the splenic red pulp where, while a site of cellular trafficking, the area is constrained enough for NO activity. Residual babesiacidal effects of NO would then be expected to be evident in blood. Despite the typically low peripheral blood parasitaemias associated with B. bovis infections, crisis forms are rarely found in stained blood films from animals experiencing severe disease resulting in death. The few infected cells that can be detected from these animals have robust looking parasites within the erythrocytes. In contrast, crisis forms are typically seen in blood films from animals that mount a successful immune response, especially young calves (16).

Mononuclear phagocytes function within a complex immunological and inflammatory milieu, and the functional response of MP is often dictated by the first regulatory cytokine to which they are exposed (20). We present evidence that the protective innate immune response of young calves to infection with B. bovis has type-1 attributes, with early induction of splenic interleukin (IL)-12 and interferon (IFN)-γ mRNA expression followed by a brief period of inducible nitric oxide synthase (iNOS) expression. In contrast, IL-12 and IFN-γ message occurred later with no detectable iNOS in the spleens of adult cattle that succumbed to the infection. In addition, the induction of IL-10 message in the spleens of adults reached higher levels of expression and was sustained longer than in calves. Finally, relative splenic transforming growth factor (TGF)-β mRNA expression levels and kinetics differed between calves and adults, suggesting a dual regulatory role for this cytokine.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experimental animals

Three Holstein-Friesian steer calves, aged 4 months, and three adult steers were maintained according to the American Association for Laboratory Animal Care procedures, and were provided an acceptable bovine ration, with water and mineral block provided ad libitum. Each animal underwent a surgical procedure at 3 months of age to marsupialize the spleen for ready access of splenic mononuclear cells (SMC) (21,22). Splenic aspirates were collected under local anethesia. Studies had previously been carried out to demonstrate that the frequency of collection had no adverse effect on the animals and no significant phenotypic or functional effect on the SMC populations (data not shown).

Experimental animal infection and sample collection

Each of the three adult and 4-month-old steers was inoculated intravenously with 105 infected erythrocytes from a stabilate of a B. bovis isolate (S1-T2Bo). The isolate is virulent, causing severe disease and high mortality in mature, fully susceptible cattle (16). Blood and spleen cell aspirates were collected several weeks prior to infection and daily thereafter. In addition, rectal temperature and haematocrit were recorded daily and differential blood leucocyte counts were made. Daily blood films were Giemsa-stained for detection of infection microscopically. Plasma from the EDTA-containing tube used to draw blood for films and haematocrit was processed to determine the daily levels of IFN-γ using an ELISA as previously described (23). Animals that developed severe disease were euthanized when in extremis.

Source and preparation of leucocytes

SMC were obtained from splenic aspirates aseptically collected in 60 ml syringes containing 15 ml acid-citrate-dextrose, pH 7·3. The splenic aspirate was processed into a single cell suspension using a tissue homogenizer, and the mixture diluted 1 : 1 in phosphate buffered saline without Ca2+ and Mg2+, pH 7·0. SMC were prepared as previously described (15). Briefly, splenic cell suspensions were layered onto 20 ml of Hypaque-Ficoll (1·086 g/l) (Accu-Paque, Accurate Chemicals, Westbury, NY, USA) and centrifuged for 30 min at 1500 g and 4°C. The interface leucocytes from similar gradients were collected, pooled, and washed in 50 ml Dulbecco's modified eagle's medium (DMEM), pH 7·2, for 7 min at 1500 g and 4°C. The pelleted cells were suspended with DMEM and centrifuged twice for 7 min at 400 g and 4°C to remove platelets. Residual erythrocytes in the pelleted cells were lysed by suspending the pellet in three volumes of sterile, ambient temperature lysis buffer (AKC: 0·15 m NH4Cl, 1·0 mm KHCO3, 0·1 mm EDTA, pH 7·3). After 1 min, the leucocytes were centrifuged for 5 min at 200 g and 4°C, washed in 50 ml DMEM, and suspended to 1 × 107 per ml in Iscove's medium (Gibco BRL, Gaithersburg, MD, USA) containing 25 mm Hepes, 2 µm glutamine, 10 µg/ml gentamicin, 50 µm mercaptoethanol, and 15% essentially endotoxin-free fetal bovine serum (< 0·06 EU/ml as assayed by limulus amebocyte lysate gelation) (Hyclone, Logan, UT, USA). Total leucocytes were plated at a density of 1 × 107 per ml in Iscoves medium.

Culture conditions

For release of nitric oxide, leucocytes were incubated in the presence of medium alone or containing stimulants as previously described (15,24) including tumour necrosis factor (TNF)-α at 2500 U/ml (Genzyme, Cambridge, MA, USA), IFN-γ at 50 U/ml (Ciba-Geigy, Basel, Switzerland through Veterinary Infectious Disease Organization, Saskatchewan, Canada), or 5 mg/ml sonicated B. bovis merozoites isolated from culture as previously described (25). Cultures were incubated for 3 days at 37°C, 5% CO2 before supernatants were assessed for evidence of NO production by detection of nitrite, the stable oxidized form of NO, using the Griess reaction.

Griess reaction

Accumulation of nitrite in culture supernatants was determined as previously described (26) by mixing 50 µl of supernatant with 200 µl of Greiss reagent (1% sulfanilamide, 0·1% napthylethylenediamine dihydrochloride, 2·5% H3PO4). The assay was incubated at room temperature for 10 min, and the absorbance at 540 nm was determined with a microplate spectrophotometer (Dynex Technologies, Chantilly, VA, USA) and compared with a standard of 1·5–200 µm NaNO2.

Reverse transcription-polymerase chain reaction (RT-PCR)

SMC were adjusted to 1 × 107 per ml when using total leucocytes, or 1 × 106 per ml when using adherent cells, and 0·1 ml used if from 96-well plates or 0·5 ml used if from 24-well plates. Samples of isolated RNA were processed for RT-PCR amplification of several cytokine mRNA as well as the house keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as previously described (24). In addition, TGF-β was also included in this study. The primer sequences used were from GenBank accession M36271 (27) (607 bp), forward 5′-CAGAAA TATAGCAACAATTCC-3′, reverse 5′-GGCTTGCGGCCC ACGTAGTACAC-3′. Briefly, RNA samples reverse transcribed by Superscript II Reverse Transcriptase (BRL) and oligo-(dT)12 primer were compared with RNA samples without reverse transcription for specific amplification of mRNA. Taq DNA polymerase (BRL) amplified products were resolved on 4% NuSieve 3 : 1 (FMC BioProducts, Rockland, ME, USA) agarose gels containing ethidium bromide. The level of mRNA expression in each sample was determined by densitometric image analysis and standardized against the GAPDH measurement (IS-1000 Digital Imaging System and Alpha-EASE 3·21 software, San Leandro, CA, USA). Each mRNA expression level is presented in relative units after normalization to the observed GAPDH expression level.

Statistical analysis

Student's t-test was used to compare maximum differences or difference in time to maximum expression between each group.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Clinical parameters during infection

As expected, the disease was more severe in the adults. Clinical parameters from both groups are shown in Table 1. The haematocrit decreased by approximately 50% in both groups, and each animal developed a febrile response between days 7 and 10 post inoculation (PI). The calves maintained a fever for 4–8 days before returning to normal. Each adult maintained a fever until just before death that occurred on days 12, 13 and 15 PI. As we have observed before, only babesial crisis forms were detected microscopically from blood films prepared from each calf, in contrast to the adults where, despite low parasitaemias, morphologically intact parasites were detected. As shown in Figure 1, both groups experienced a reduction in circulating leucocytes, primarily lymphocytes, but while the numbers continued to decrease until death in the adults, there was a return to normal numbers in the calves and, by day 12 PI, there was a significant difference (P < 0·05). In the calves, lymphocytes continued to increase beyond day 12 and reached lymphocytotic levels for 2–3 days (data not shown). Monocyte numbers fluctuated, but there was no significant difference between the two groups, and no dramatic individual change in numbers except for one animal whose counts more than doubled on day 10 PI.

Table 1.  Clinical parameters during the response of adult steers and young calves to an initial infection with Babesia bovis
AnimalsHematocritaOnset and (duration) of feverParasitemiabStatusc
  • a

    Lowest value (percent reduction from preinfection level).

  • b

    b CF, Crisis forms in calves (days that were detected); percent infected erythrocytes in adults (days that were detected).

  • c

    c S, survived; D, death (day of death).

Calves
 C-107715 (57%)Day 8 PI (8 days)CF < 0·1% (days 13, 14, 15 PI)S
 C-107815 (56%)Day 7 PI (8 days)CF < 0·1% (days 11, 12 PI)S
 C-107919 (39%)Day 10 PI (4 days)CF < 0·1% (days 12, 13 PI)S
Adults
 C-77415 (56%)Day 9 PI (6 days)0·1% (day 14 PI)D (day 15 PI)
 C-77524 (33%)Day 7 PI (5 days)0·27, 0·4% (days 10, 11 PI)D (day 12 PI)
 C-77615 (57%)Day 7 PI (6 days)0·1% (day 12 PI)D (day 13 PI)
image

Figure 1. Total and differential blood leucocyte counts were determined throughout infection. Showing (a) the total leucocyte counts, (b) the lymphocyte counts and (c) the monocyte counts for calves (◆) and adults (▪). Maximum differences at day 12 PI were significant (P < 0·05).

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Kinetics of IL-12, IFN-γ and iNOS

Due to the importance of the spleen in the response to infection, the focus of the analyses was on spleen cell preparations. RNA was extracted from SMC throughout the reponse to infection and the mRNA expression of various cytokines and iNOS were determined. The induction of splenic IL-12 and IFN-γ message from calves increased soon after experimental inoculation, 4–5 days before a similar response was seen in adults (Figure 2). In calves, the IL-12 message peaked between days 3 and 4 PI followed by the peak of IFN-γ mRNA expression between day 5 and 6 PI. In adults, the peak splenic mRNA expression of IL-12 and IFN-γ occurred between days 6 and 7 and days 9 and 10 PI, respectively. iNOS message was detected in SMC from calves on days 7 and 8 PI (Figure 2a), immediately following the peak of splenic IFN-γ message. No iNOS message was detected in SMC from adults.

image

Figure 2. Kinetics of IFN-γ mRNA expression relative to IL-12 expression in SMC from (a) calves and (b) adults during infection. IL-12 = black bars and IFN-γ = white bars. The arrows in (a) indicate the days of iNOS expression in SMC from calves. The time to reach maximum expression was significantly different between the two groups: IL-12 (P < 0·1) and IFN-γ (P < 0·05). IFN-γ expression levels were also significantly different on days 13 and 14 (P < 0·05).

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Plasma IFN-γ levels reflected the disparity in mRNA kinetics. Plasma levels of IFN-γ increased significantly sooner (P < 0·05) in calves, reaching maximum levels on day 6 PI compared to maximum levels between days 11 and 13 in adults (Figure 3).

image

Figure 3. Kinetics and levels of IFN-γ produced and detected in plasma from calves (white bars) and adults (black bars). The time to reach maximun IFN-γ levels in the plasma was significantly different between the groups (P < 0·05).

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IL-10 message in relation to IFN-γ and TNF-α message and the expression of TGF-β mRNA

Splenic IL-10 message was induced in SMC from calves and adults at approximately the same time. However, while not statistically significant, the overall expression levels in SMC from adults was 30% greater and remained elevated longer (until death) (Figure 4). As the IL-10 expression level increased, both IFN-γ and TNF-α expression decreased in the spleens of both calves and adults. The adult TNF-α level decreased below that of the preinoculation level. IL-10 message also decreased after peak expression and, in calves, diminished to near preinoculation levels. TGF-β mRNA was expressed from SMC of each animal throughout the experimental period. However, the expression level from calves was lower than from adults prior to, and during, the first 6 days of infection. Between days 6 and 7 PI, the expression level from the SMC of calves increased by approximately 50% and exceeded that of SMC from adults (Figure 4c). The difference in expression levels was statistically significant for days 3 and 7–10 PI (P < 0·05).

image

Figure 4. IFN-γ and TNF-α mRNA expression relative to IL-10 expression in SMC from (a) calves and (b) adults during infection. IFN-γ = shaded bars, TNF-α = white bars and IL-10 = black bars. The time to reach maximum expression for IFN-γ and TNF-α was significantly different between the two groups (P < 0·05). TGF-β mRNA expression in SMC from calves (white bars) and adults (black bars) during infection (c). The time to maximum expression was significant between the two groups (P < 0·05).

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Nitric oxide production from SMC during infection

Bovine MP can respond to IFN-γ plus TNF-α as soluble signals for the production of NO for a short time in culture and early in the differentiation from monocytes to macrophages. These monocytes are able to respond to exogenous TNF-α alone if the cells are either simultaneously, or recently exposed to IFN-γ(24). MP can also produce NO in response to IFN-γ alone if TNF-α is being produced by MP. Therefore, NO production resulting from stimulation with IFN-γ alone serves as a biological assay for the presence of functional TNF-α(28). NO is induced when bovine MP are exposed to B. bovis merozoites in the presence of IFN-γ(29) and TNF-α(28). This provided a means to examine directly ex vivo, the condition of MP from each animal with respect to NO production. As shown in Figure 5, splenic MP became responsive to exogenous IFN-γ, TNF-α or merozoites by day 4 PI in calves but was delayed until day 6 PI in adults. NO levels produced by the same number of cells were nearly three-fold greater in calves compared to adults on day 7 PI (the peak of response to merozoites) and a six-fold increase above preinfection levels. NO levels produced by MP in response to stimulation with TNF-α remained relatively high throughout the course of the acute infection in both groups (although significantly greater from calves, P < 0·01 and P < 0·05) indicating that IFN-γ was present. Significantly greater levels of NO were produced by merozoite-stimulated MP from calves (P < 0·01). However, in contrast to TNF-α stimulated MP, the reponse to B. bovis merozoites decreased precipitously after day 7 PI in both groups, suggesting that the IFN-γ-dependent production of NO was being downregulated even in the continued presence of IFN-γ. Figure 5 also shows the NO response of the MP to IFN-γ alone as stimulant, a measure of the functional level of TNF-α. Again, there was a NO response to IFN-γ from all animals but it occurred earlier and of significantly greater magnitude in calves (P < 0·01). The NO response to IFN-γ decreased in parallel with the NO response to merozoites in the adults (Figure 5b), where it remained robust in calves (Figure 5a). The decline in NO response to merozoites or IFN-γ alone followed the increase in IL-10 mRNA expression providing evidence for the downregulation of TNF-α, IFN-γ and iNOS by IL-10 that was more pronounced in adults than calves.

image

Figure 5. Ability of SMC taken during infection from (a) calves and (b) adults to respond to stimulation in vitro with merozoites (shaded bars), TNF-α (white bars) or IFN-γ (black bars) with the production of nitric oxide. Nitrite levels as a surrogate for nitric oxide were measured from culture supernatants as described in the Materials and Methods. Significantly more NO was produced by merozoite stimulated MP from calves on days 4–9 (P < 0·01) (except day 6, P < 0·05). Significantly more NO was produced by TNF-α stimulated MP from calves on days 4, 7, and 8 (P < 0·01) and days 5 and 9 (P < 0·05). Significantly more NO was produced by IFN-γ stimulated MP from calves on day 4–10 (P < 0·01).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results of this study are consistent with the hypothesis that NO plays a role in controlling B. bovis infection in naive young calves during an innate, type-1 response ocurring in the spleen. There was also evidence that NO may contribute to the regulation of the type-1 response. While the focus was on spleen cells, peripheral blood mononuclear cells (PBMC) from these animals yielded results similar to those obtained with SMC. The innate immunity was sufficient to prevent severe disease in calves, in contrast to that of adult animals that succumbed to the same challenge with B. bovis. The two prominent features distinguishing the response to initial infection between the groups were the induction of an early and vigorous splenic IL-12 mRNA response in calves that elicited an earlier IFN-γ response than in adults, and the induction of iNOS in the spleen of calves that was not apparent in adults.

The production of IFN-γ is important in both innate and acquired immunity for activating MP and regulating the switch to the opsonic immunoglobulin isotype immunoglobulin G2(30). In both situations, IL-12 plays a role in upregulating the production of IFN-γ(31,32). In addition, IL-12 has been shown to suppress the expression of IL-10 in B. bovis-specific T-cell clones (33). The infection, and perhaps the parasite itself, is responsible for the induction of IL-12 in MP. Exposure to merozoites in vitro, results in expression of IL-12 mRNA (28,29). This has also been documented in mice with Toxoplasma gondii where, following MP exposure to tachyzoites, both IL-12 and TNF-α are induced, which in turn triggers the production of IFN-γ by NK cells (34,35).

The reason for the disparate appearance of IL-12 mRNA in the spleen of calves and adults is not clear. However, the early induction was associated with the protection of the calves. IL-10 has been demonstrated to downregulate gene expression of bovine IL-12 as well as IFN-γ, TNF-α, IL-1, IL-2, IL-6 and iNOS (5,24,36). Most of the immunosuppressive effects of IL-10 are due to interactions with MP, either inhibiting effector cell functions, as suggested in this study and others (37), or by disrupting T-cell functions indirectly by inhibiting the interaction of T-cells with MP as accessory cells (5,38,39). The induction of IL-10 message in the spleen of both calves and adults was followed by reduced expression levels of IL-12, IFN-γ and TNF-α. In addition, the IL-10 mRNA expression levels were greater in SMC from adults and remained prominent longer. IL-10 induction kinetics were not statistically different between the calves and adults. Therefore, it is unlikely that IL-10 influenced the IL-12 induction kinetics. However, IL-12 induction in the spleen of calves did occur in the absence of IL-10 while, in adult spleens, IL-12 was induced in the context of IL-10. This may be a key to the successful response in calves.

The role of TGF-β in controlling parasitaemia and preventing inflammatory response pathology during malaria has recently been documented (40). In these studies, both the kinetics and levels of TGF-β were important. High levels inhibited type-1 immune responses and, if produced too early, prevented IFN-γ-and TNF-α-mediated control of infection. Increased TGF-β levels later during infection served to control overproduction of inflammatory cytokines, and were demonstrated to downregulate TNF-α production. Upregulation of IL-10 by TGF-β was also demonstrated and occurred without a corresponding decrease in IFN-γ(40). The results of our study are compatible with these observations and, although TGF-β was not assayed directly, mRNA expression levels suggest that a similar effect may exist in the spleen of cattle.

Bovine MP require TNF-α as a costimulant for the induction of iNOS (15). As a costimulator, TNF-α functions in an autocrine fashion and is so easily induced in monocytes that it has the appearance of constituitive expression. In contrast, fully differentiated macrophages require purposeful stimulation to affect induction. In the presence of IFN-γ, TNF-α mRNA is upregulated in monocytes and monocyte-derived macrophages exposed to B. bovis merozoites (28,29). Thus, in addition to activated macrophages (and perhaps functionally more important) are the monocytes that traffic through the spleen, make TNF-α and, together with local IFN-γ, trigger iNOS.

TNF-α mRNA expression was evident throughout the infection in the spleen of both calves and adults, although there was evidence for an increase early during infection followed by a decrease in conjunction with the presence of IL-10. While the evidence indicates that there was ample TNF-α present at the onset of the immune response in both groups, the level of mRNA expression in adults dropped below what may be a critical level for TNF-α production; thus, prematurely arresting its contribution as a costimulant for iNOS signal transduction, a condition reflected in the NO response to stimulation of SMC with IFN-γ alone. This event coincided with the onset and prominent expression of IL-10 message in adult spleens. In addition, iNOS and NO production resulting from in vitro stimulation of bovine monocytes with B. bovis merozoites and IFN-γ and autocrine TNF-α are completely inhibited in the presence of IL-10 (28).

NO is a free radical and thus extremely reactive. It not only has antimicrobial activity, but it can also be damaging to host cells (41,42). Prolonged production of IFN-γ and TNF-α also contributes to pathology (39). Thus, production of NO and inflammatory cytokines must be tightly regulated. This may be directed by antagonistic cytokines, such as IL-10, or through an autoregulatory NO pathway. NO has been shown to suppress lymphocyte proliferation (43,44) and has been demonstrated to actually decrease the production of IFN-γ in T-cell clones (45). Interestingly, no iNOS message was detected in the spleens of the adults and IFN-γ mRNA expression was actually higher than in calves. In addition, NO has been shown to increase IL-4 rather than decrease IFN-γ, indicating that NO may act in a feedback inhibition circuit to limit the overproduction of NO and inflammatory cytokines (46). IL-4 has been shown to be an inhibitor of bovine iNOS, unless in the presence of IFN-γ, where the effects were reversed (47). IL-4 message was never detected in the spleens or PBMC from any of the animals in our study. Another possible means by which NO could regulate itself and other inflammatory cytokines is through the modulation of IL-12. Biologically active IL-12 is heterodimeric, consisting of p40 and p35 subunits (48–50). Homodimeric p40 production can interfere with normal biological activity by competing for the IL-12 receptor, resulting in consequent suppression of type-1 and natural immunity (51–53). NO has been demonstrated to upregulate p40 subunit gene expression (54). The p40 subunit mRNA expression was the only one monitored in our study, thus it can only be speculated that p40 production, in association with p35, participated in the early protective response and that subsequent production of homodimeric p40 may have contributed to the regulation of the entire inflammatory response.

It has been reported that the iNOS inhibitor, aminoguanidine (AG) administered to 6–8 month-old calves at the time of intravenous inoculation of B. bovis, resulted in less severe disease as measured by haematocrit and pyrexia (55). The authors attributed this to the immunomodulatory effects of NO. However, NO was not monitored during the experiment, and detectable levels of AG were absent by day 6, a day before iNOS was demonstrated in the 4-month-old calves of our study. In their study, it is possible that NO was produced at day 7 PI when both the AG and control group parasitaemias began to decline. It is possible that the reduced fever and severity of anaemia was due to some other effect of AG or that NO kinetics were different in their study compared to ours. It is also possible that AG inhibition of serum but not intracellular polyamine oxidases allowed for the acculmulation of known babesiacidal compounds within the erythrocytes (56). NO does have immunomodulatory attributes and downregulates type-1 responses transiently as a means of preventing excessive inflammatory cytokine production (57). Thus, it is likely that it plays a short-term, but critical and dichotomous role in the response to initial B. bovis infection.

The results of this study suggest that if IL-12 and IFN-γ are produced prior to IL-10, the innate immune response will be protective. In contrast, if IL-10 is influential first, the type-1 response will be either delayed or inhibited until it is too late to arrest the disease process. In addition to the modulation of NO production, the eventual presence of IL-10 may play a role in controlling inflammatory cytokines and also a role in enhancing other MP effector functions, such as phagocytosis, through the upregulation of complement and Fc receptors (58,59). The pattern and sequence of induction of cytokine mRNA apparent in this study suggests that immunization strategies should consider methods to incorporate or modulate the production of IL-12 to elicit the desired immune response.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We wish to thank Pete Steiner, Duane Chandler and Robert Finch for excellent animal care, Paul Lacy for technical assistance and Shirley Sandoval and Emma Karel for assistance in surgery and tissue collection. This study was supported by USDA-ARS-ADRU-CRIS # 5348-32000-010.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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