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Infection and Immunity, September 2000, p. 5393-5400, Vol. 68, No. 9
TB Research Group1 and
TB Diagnostics Laboratory,2 Veterinary
Laboratories Agency, Addlestone, Surrey KT15 3NB, and Liverpool
School of Tropical Medicine, Pembroke Place, Liverpool L3
5QA,3 United Kingdom
Received 9 March 2000/Returned for modification 7 April
2000/Accepted 14 June 2000
This study shows that gamma interferon (IFN- Tuberculosis is a major public
health problem worldwide and a leading cause of mortality due to
infectious disease (28). The emergence of drug-resistant
strains of Mycobacterium tuberculosis (24) and
the potential effects of the increasing prevalence of human
immunodeficiency virus and AIDS (22) makes the search for
both drug and vaccine solutions all the more pertinent. The incidence
of Mycobacterium bovis tuberculosis in British cattle is
also increasing, and the urgency for new and improved cattle vaccines
and diagnostic aids has been clearly stated in a recent independent
scientific review (16).
For the rational development of such reagents, it is important to
assess the nature of the immune responses that occur following infection. In tuberculosis, much work has concentrated upon the two
cytokines, gamma interferon (IFN- Cytokine responses in human tuberculosis appear to be more complex.
Data reported from some studies have reflected those gathered from
mouse models with a similar, if not quite so polarized,
type-1-to-type-2 cytokine shift from mild through to advanced clinical
disease (1, 8). However, others have observed an increase in
serum IFN- Opinions are similarly divided upon whether polarized T-cell responses
occur against infectious agents in cattle. Brown et al. (3)
described the presence of both IFN- This study describes the IL-4 and IFN- Mycobacterial antigens.
Tuberculin PPD preparations from
M. bovis (PPD-M) and from Mycobacterium avium
(PPD-A) were produced at the Veterinary Laboratories Agency (VLA) of
Weybridge (VLA Weybridge) as described previously (36).
Recombinant ESAT-6 (36) was kindly provided by Adam Whelan
(VLA Weybridge).
Animals. (i) Experimental M. bovis infection.
Six female Friesian-Limousin cross-calves (CN1101, CN1104, CN1100,
CN1104, CN1098, and CN1109) approximately 6 months old were obtained
from a herd free of M. bovis infection, as defined by a
history of negative skin test results. These animals were also found to
be negative in an IFN- (ii) Naturally infected M. bovis field reactors.
Blood samples were obtained from 21 skin test-positive
(SCITT+) cattle. Where gross lesions were visible, lesioned
material was collected for microbiological culture. In the absence of
visible lesions, a standard pool of lymph node material (cranial,
bronchial mediastinal, and mesenteric lymph nodes) was collected at the abattoir for the bacterial culture which was used to confirm the presence of M. bovis.
(iii) Uninfected controls.
Thirteen cattle were obtained
from farms free of M. bovis (confirmed as above by a history
of negative skin test [SCITT (iv) Experimental helminth infection.
Six Jersey bull calves
reared off pasture, with no prior exposure to parasitic helminths, were
infected with the filarial nematode Onchocerca ochengi.
Infective larvae were produced in blackflies as described elsewhere
(2) using cryopreserved microfilariae collected from cattle
in northern Cameroon. Animals were infected at approximately 6 months
of age and received 350 infective (third-stage) larvae, each by
subcutaneous injection along the ventral midline. Patent infections
(detected by the appearance of microfilariae in skin biopsies from the
ventral midline) developed after 9 to 15 months. One uninfected control
animal was used in the helminth study for longitudinal comparisons.
Peripheral blood mononuclear cells (PBMC) culture supernatants from
blood taken at the same time point as that for O. ochengi-infected cattle in our study were negative for both
IFN- PBMC culture.
PBMC culture supernatants were assessed for
IL-4 and IFN- Whole-blood culture for IFN- Cytokine assays. (i) IFN- (ii) IL-4.
IL-4 activity in neat PBMC culture supernatants
was measured by using a B-cell bioassay according to the method of
Kuhnle et al. (17). Briefly, bovine B cells from a
SCITT Pathological findings of M. bovis-infected cattle. (i)
Experimental infection.
Positive SCITT responses ( (ii) Field reactors.
Data concerning the naturally infected
field reactor cattle are shown in Table
1. Visible lesions were observed in 17 of 21 animals, while 4 of 21 were classed as nonvisible-lesion reactors. However, M. bovis infection was confirmed in all animals by
microbiological culture of M. bovis from tissues collected
postmortem. Skin test response results (
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Distinct Response Kinetics of Gamma Interferon and
Interleukin-4 in Bovine Tuberculosis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and interleukin-4
(IL-4) cytokine responses are produced by peripheral blood cells in
cattle infected with Mycobacterium bovis. The different kinetics of the IFN- and IL-4 responses to bovine tuberculin and to
ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN- was observed that was maintained throughout the period studied. In contrast, the
IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a
lower dose of M. bovis which did not result in the
development of lesions, positive DTH skin test, or substantial IFN-
responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors
showed increased IFN- and IL-4 responses compared to uninfected
cattle and that both of these cytokines were equally able to
differentiate infected from uninfected animals. The magnitude of the
M. bovis-induced IL-4 responses were found to be similar to
the antigen-specific IL-4 responses of cattle infected with the
parasitic nematode Onchocerca ochengi, further supporting
the presence of this type 2 cytokine in bovine tuberculosis.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and interleukin-4 (IL-4), and the
inferred polarization of T-cell subsets. Mouse models of tuberculosis
have been shown to display polarized T-cell responses, where IFN- is
clearly an important cytokine in the control of the infection
(12), largely due to its potent role in macrophage activation (7). Conversely, IL-4 has been reported to be
associated with the development and progression of disease
(14), although other studies using IL-4 gene-disrupted mice
failed to support such a role for IL-4 (10, 23). It has also
been postulated that the reactivation of latent tuberculosis involves a
type-1-to-type-2 cytokine shift (13).
(35) and in local IFN- and IL-4 responses
of bronchoalveolar lavage cells (32, 34) in patients with
active tuberculosis, suggesting the presence of both type 1 and type 2 cytokine responses during disease.
and IL-4 mRNA in the T-cell
clones of cattle infected with the parasitic helminth Fasciola
hepatica. In contrast, bovine diarrhea virus in cattle has
recently been shown to induce different T-cell subsets
(CD4+ and CD8+) to produce IL-4 and IFN-
proteins, respectively (29). In bovine tuberculosis, IFN-
responses are well known to be produced after infection (9, 21,
39, 40), but IL-4 responses during infection have not yet been investigated.
responses in the peripheral
blood of cattle both experimentally and naturally infected with
M. bovis. Cytokine responses to tuberculin purified protein derivative (PPD) and to a recombinant form of the immunodominant M. bovis antigen ESAT-6 (25, 26) were measured.
The response kinetics of these two cytokines in experimentally infected
cattle and the ability of both IFN- and IL-4 to differentiate
between infected and uninfected animals are discussed.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
diagnostic assay for bovine tuberculosis. Two
of the calves (CN1101 and CN1107) were infected with 6.6 × 103 CFU, two calves (CN1100 and CN1104) were infected with
6.6 × 104 CFU, and two calves were infected with
6.6 × 105 CFU M. bovis intratracheally
according to the method of Buddle et al. (4). During the
study, animals were housed in a high-security isolation unit under
negative pressure, and expelled air was filtered. Two weeks prior to
the postmortem point, the single comparative intradermal tuberculin
skin test (SCITT) was performed on all animals as described previously
(11). Euthanasia was carried out at 20 weeks postinfection
by intravenous injection of sodium pentobarbitone, and a detailed
postmortem analysis was performed as described below. For lymph nodes,
each of the following types of lymph nodes (or lymph node chain) was
removed aseptically: right and left lateral retropharyngeal, right and
left medial retropharyngeal, right and left submandibular, right and
left cervical superficial, right and left bronchial, cranial
mediastinal chain, caudal mediastinal chain, and mesenteric. These
lymph nodes were subsequently serially sliced (ca. 2 mm) with a scalpel
and inspected. Samples from individual nodes and lesions were obtained for mycobacterial culture and histological examination. The remaining superficial and visceral lymph nodes were inspected in situ. For lungs,
each lobe was serially sliced (ca. 5 mm) with a sharp knife. All slices
were palpated and inspected. The respiratory airways were cut open as
far into the lung parenchyma as possible and then inspected.
] results). These animals
represented the control negative group for this investigation.
and IL-4 responses (data not shown).
responses (field reactors, negative controls, and
helminth-infected animals) or for IL-4 responses alone (experimental
M. bovis-infected animals). PBMC were separated from
heparinized venous blood over Histopaque 1077 (Sigma) and resuspended
in culture medium (RPMI 1640 with Glutamax [Gibco] supplemented with
5% CPSR [serum replacement; Gibco], nonessential amino acids
[Gibco], 100 U of penicillin per ml, 100 µg of streptomycin per ml,
and 5 × 105 M 2-mercaptoethanol [Gibco]). Cells
were resuspended to 2 × 106/ml, and 0.1 ml was added
per well to 96-well flat-bottom microtiter plates (Nunc). Antigens were
added, at 0.1 ml/well in triplicate (all at a 10-µg/ml final
concentration). The cultures were incubated for 6 days at 37°C in 5%
CO2. Supernatants (100 µl/well) were then harvested and
assayed for IFN- and IL-4. PBMC from helminth (O. ochengi)-infected cattle, obtained 27 days postinfection, were
cultured under similar conditions, except that the medium was
supplemented with 10% fetal calf serum (FCS) in place of CPSR, parasite antigen (derived from a phosphate-buffered saline extract of
adult worms) was used at 50 µg/ml, and cells were cultured for 3 days
at 5 × 106/ml. The incubation times of PBMC from
M. bovis and O. ochengi-infected cattle (6 and 3 days, respectively) for supernatants were set according to the optimal
PBMC proliferation times determined for these two different
experimental infection systems. Supernatants were then harvested and
assessed for IFN- and IL-4. Data are shown for cytokine activity at
27 days postinfection when both IL-4 and IFN- were observed at peak
response levels (S. Graham et al., unpublished data).
.
IFN- production by
experimentally infected animals was assessed using the whole-blood
culture method described by Emery et al. (9). Peripheral
whole blood was diluted 1:1 in culture medium (120 µl of blood plus
120 µl of medium per well in duplicate in 96-well flat-bottom tissue
culture trays) in the presence or absence of antigen (10 µg/ml, final
concentration) for 24 h. Supernatants were then harvested and
assayed for IFN-.
.
Whole-blood and PBMC neat
supernatants were assessed for IFN- content using a commercially
available antigen capture enzyme-linked immunosorbent assay (ELISA)
kit, Bovigam (CSL, Ltd., Parkville, Victoria, Australia), according to
the manufacturer's instructions. Results are expressed as changes in
the optical density (
OD; i.e., the OD at 450 nm
[OD450] is the mean OD of antigen-stimulated supernatants minus the mean OD of the control, unstimulated
supernatants at 450 nm). A positive result was taken as an
OD450 of antigen-stimulated supernatant that was greater
than twice the OD450 of the control, unstimulated
supernatant (36) and also greater than 0.2.
animal were positively sorted from PBMC using the
monoclonal antibody IL-A58 (immunoglobulin G2a [IgG2a]; courtesy of
the Institute for Animal Health [IAH], Compton, Berkshire, United
Kingdom) specific for bovine immunoglobulin light chains
(38), which both labels and preactivates the B cells by
cross-linking the surface immunoglobulin. PBMC were then incubated with
rat anti-mouse IgG2(a+b)-coated microbeads before positive sorting
using the MACS column separation system (Miltenyi Biotech, Surrey,
United Kingdom). B cells (determined to be >97% pure by
fluorescence-activated cell sorter analysis) were eluted, washed, and
resuspended to 106/ml in tissue culture medium (RPMI 1640 with Glutamax [Gibco] supplemented with 10% FCS [Gibco],
nonessential amino acids [Gibco], 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 5 × 105 M
2-mercaptoethanol [Gibco]), and 100 µl was added per well to 96-well tissue culture trays with 50 µl of PBMC supernatants in duplicate. Plates were incubated for 24 h at 37°C in 5%
CO2, pulsed with tritiated thymidine, and incubated for a
further 24 h before harvesting. The results are presented as
stimulation indices (SI), i.e., the mean counts per minute (cpm) of
B-cell proliferation in the presence of antigen-stimulated supernatant
divided by the mean cpm of B-cell proliferation in the presence of
unstimulated control supernatant. A positive response was taken as an
SI value of >3. The specificity of this bioassay for IL-4 has been
previously determined by inhibition with a neutralizing monoclonal
antibody (29). However, a neutralizing monoclonal antibody
to IL-4 (IAH) was included in a number of the assays in this report and
equally neutralized both the recombinant IL-4 (IAH) used as a positive control in all assays and the IL-4 activity present in the supernatants tested (data not shown).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
SCITT from 10 to 12 mm) were observed in four experimentally infected cattle (CN1100,
CN1104, CN1098, and CN1109). These four animals had received infection
doses of M. bovis of 6.6 × 104 or 6.6 × 105 CFU. At the postmortem point, all four animals
presented with gross tuberculous lesions typical of natural bovine
tuberculosis: CN1109 in the lateral and medial retropharyngeal, right
submandibular, and bronchial lymph nodes; CN1098 in the medial
retropharyngeal, bronchial, and mediastinal lymph nodes; CN1104 in the
lateral and medial retropharyngeal, submandibular, bronchial, and
mediastinal lymph nodes; and CN1100 in the lateral retropharyngeal,
bronchial, mediastinal, and mesenteric lymph nodes and in the lungs.
The lesions were histopathologically confirmed as tuberculous
granulomas, and M. bovis was cultured from these lesions.
The two animals (CN1101 and CN1107) that received the lowest dose
(6.6 × 103 CFU) of M. bovis remained
SCITT. No lesions were observed in these animals, and
M. bovis was not confirmed either by histopathology or by
microbiological culture of tissues.
SCITT) ranged from 2 to 42 mm.
TABLE 1.
M. bovis
field reactorsa
IFN- and IL-4 responses following experimental infection with
M. bovis.
Figure 1 shows the
IFN- and IL-4 responses induced by both PPD-M and ESAT-6 in the four
SCITT+ experimentally infected cattle (CN1100, CN1104,
CN1098, and CN1109). Increased IFN- and IL-4 responses were observed
in all four animals, and cytokine responses to ESAT-6 largely reflected
the responses to PPD-M, although they were slightly lower in most
cases.
|
) and ESAT-6
(
) are shown for each time point. Results are expressed as the
response to either PPD-M and/or ESAT-6 was
observed in all four animals by 4 weeks postinfection, as previously described (31). In general, these responses continued to
increase up to 6 to 8 weeks postinfection. Strong positive responses,
with some variation over time, were then sustained throughout the
period studied in all animals.
Increased IL-4 activity induced by tuberculin was detected slightly
later than the IFN- responses, with a sharp rise occurring at around
6 to 8 weeks in all animals. In contrast to the IFN- responses,
which were sustained during the period studied, IL-4 activity was
contained within a distinct peak over a short period of time (6 to 8 weeks). A second peak of IL-4 activity was observed in response to
ESAT-6 at 16 weeks in one animal only (CN1104), and this peak again
lasted for approximately 6 weeks. The early peak of ESAT-6-induced IL-4
production (observed in CN1109, CN1098, and CN1104) was not observed in
CN1100, where instead we observed ESAT-6-stimulated IL-4 production at
16 to 20 weeks postinfection.
IFN- and IL-4 responses in naturally infected field
reactors.
Figure 2 shows the IFN-
and IL-4 responses observed in PBMC supernatants from naturally
infected (SCITT+) and control uninfected
(SCITT) cattle following in vitro stimulation with PPD-A,
PPD-M, and ESAT-6. The infected nonvisible-lesion animals were grouped
together with the infected visible-lesion animals since there was no
obvious difference between these groups on the basis of IFN- or IL-4 production.
|
, positive responses; 
, negative responses).
and IL-4
responses to PPD-A were identified in 15 and 14 of 21 cattle,
respectively, while in the control uninfected (SCITT)
group 5 of 11 animals showed positive IFN- responses, and 3 of 11 showed positive IL-4 responses to PPD-A. In the responses to PPD-M,
positive IFN- and IL-4 responses were observed in 20 of 21 SCITT+ cattle each, while in the SCITT group
only 2 and 1 of 11 animals gave positive IFN- and IL-4 responses,
respectively. In all SCITT+ animals that gave positive
cytokine responses, the IFN- or IL-4 response to PPD-M was always
higher than the response to PPD-A in individual animals. This finding
was in keeping with the normal method of diagnostic practice (i.e.,
responses to PPD-M to exceed responses to PPD-A). Of 21 SCITT+ cattle, 11 gave positive IFN- and IL-4 responses
to ESAT-6, representing 55% of those animals that responded to PPD-M.
None of the SCITT cattle gave positive IFN- or IL-4
responses to ESAT-6. No correlations were observed between the
magnitude of IFN- responses and the magnitude of IL-4 responses to
individual antigens within the SCITT+ group or between the
size of the SCITT response and the magnitude of the cytokine responses.
IFN- and IL-4 responses in low dose M. bovis-challenged calves.
Figure
3 shows the IFN- and IL-4 responses of
the two animals (CN1101 and CN1107) that were challenged
intratracheally with the lowest dose (6.6 × 103 CFU)
of M. bovis. These animals showed a negative SCITT test at
20 weeks postchallenge. No lesions were observed postmortem, and tissue
samples removed for microbiological culture were negative for M. bovis. The results show that a weak and transitional positive IFN- response to PPD-M was observed in one animal (CN1101) at 8 weeks postchallenge. No other positive IFN- responses to any other
antigens tested (MPB64, MPB70, MPB83, Ag85, hsp16.1, hsp65, hsp70, and
38kDa; data not shown) were recorded in these animals. In contrast,
substantial IL-4 responses to both PPD-M and ESAT-6 were observed in
both animals from 10 to 12 weeks postchallenge. IL-4 responses to PPD-M
in both animals peaked at 16 weeks before a decline toward 20 weeks
postchallenge. IL-4 responses to ESAT-6 again reflected those to PPD-M,
except in animal CN1107, where the peak IL-4 activity occurred at 12 weeks, and this response was maintained over the 20 weeks of the
experiment.
|
Comparison of M. bovis-induced cytokines with those
induced by a parasitic helminth.
The IL-4 responses of M. bovis-infected cattle were compared to those observed in an
IL-4-inducing bovine model of helminthic infection. We used the same
B-cell bioassay to measure the helminth-specific IL-4 responses of
cattle experimentally infected with the parasitic nematode O. ochengi. Figure 4 shows these
helminth-specific IFN- and IL-4 responses at a single time point 27 days postinfection, when maximal cytokine responses were obtained
(Graham et al., unpublished data).
|
, positive responses;
, negative responses).
induced by M. bovis and
O. ochengi infections were also comparable, with all 6 O. ochengi-infected animals generating positive
antigen-specific IFN- responses (OD450, 0.31 to 1.21)
compared to 20 of 21 M. bovis-infected animals generating PPD-M-specific responses (OD450, 0.46 to 1.56).
| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study describes the presence of specific IL-4 responses in
experimental and naturally acquired bovine tuberculosis and compares
these IL-4 responses with the IFN- responses that are well known to
occur during M. bovis infection (4, 9, 21, 25, 26, 30,
31, 30, 40). The antigens used in this study were the bovine and
avian tuberculin preparations that are routinely used in the diagnosis
of bovine tuberculosis, along with a recombinant form of ESAT-6. The
ESAT-6 antigen occurs in pathogenic but not in most nonpathogenic
mycobacteria (33), is a dominant antigen recognized by T
cells following M. bovis infection of cattle (25, 26,
30, 31), and has shown potential in the development of
more-specific diagnostic assays for bovine tuberculosis (5, 25,
36).
The IFN- test is a well-established method for the diagnosis of
bovine tuberculosis (40) and has been used in parallel with
the tuberculin skin test, with a sensitivity of 95.2% in the
successful Australian Tuberculosis Eradication Campaign
(39). The IFN- test has also been demonstrated to
identify naturally infected animals that present as SCITT
(21). In our study of experimental M. bovis
infection, IFN- responses to tuberculin and ESAT-6 were detected
from 4 weeks postinfection, and these responses were sustained
throughout the period studied. Positive IFN- responses were also
observed in 20 of 21 naturally infected field reactor cattle.
IL-4 activity was measured using a sensitive bioassay. The specificity
of this assay has been confirmed previously using a neutralizing
monoclonal antibody (29). Using this assay we have demonstrated the presence of IL-4 activity in cattle both
experimentally and naturally infected with M. bovis. IL-4
responses in experimentally infected animals were detected
approximately 2 weeks after the onset of positive IFN- responses,
peaked noticeably at 8 weeks postinfection, and then rapidly declined
and generally remained low. One exception to this was animal CN1104, in
which a second IL-4 peak was observed against ESAT-6 at 16 weeks.
The observed kinetics of IFN- and IL-4 responses in bovine
tuberculosis allow for some comment on the possible mechanism of
cytokine interplay that may be in operation. IFN- is a dominant proinflammatory cytokine during tuberculosis infection. One of the
major roles of IFN- is to activate macrophages which then produce an
array of proinflammatory cytokines, including IL-1
and TNF-
, both
of which are thought to play an important part in the control of
mycobacterial infection (15, 37). The delayed IL-4 response
in experimental cattle relative to the IFN- response may be
representative of an anti-inflammatory response which acts to dampen
the IFN--driven inflammation. Interestingly, in all animals a
decrease in IFN- activity was preceded by a peak activity in IL-4.
In support of an anti-inflammatory role for IL-4 in tuberculosis, recent reports by Mendez-Samperio et al. (19, 20) have
demonstrated the downmodulation of mycobacterial antigen-induced
IL-1 responses in the peripheral blood of BCG-vaccinated individuals
by IL-4. Interestingly, the IL-4 responses observed in our
experimentally infected cattle were not sustained and decreased
rapidly, leaving the IFN- response in place at a time when
tuberculous lesions would be developing. These results therefore
demonstrate the development of tuberculous lesions in cattle in the
presence of high IFN- and reduced or absent IL-4 responses and
suggest that in cattle an unchecked inflammatory IFN- response may
be contributing to the development of lesions.
Experimental cattle challenged with the lowest dose (6.6 × 103 CFU) of M. bovis did not show the
characteristic IFN- responses seen in the higher-dose (6.6 × 104 or 6.6 × 105 CFU) cattle but did show
equivalent IL-4 responses. Different cytokine responses to different
doses of mycobacteria have been reported in mice infected with M. bovis BCG (27). In that case, however, the low-dose BCG
was found to stimulate an exclusively type 1 response, while the higher
dose induced a type 1-type 2 mixed response, which is the opposite of
our findings in cattle. A possible interpretation for these
observations is that the IL-4 response may be indicative of a cryptic
infection following low-dose exposure that involves mainly local
inflammatory responses. If this were true, then the measurement of
specific IL-4 responses could provide additional information about the
exposure of cattle to M. bovis.
The IL-4 responses observed in naturally infected field reactors
distinguished infected from noninfected animals. For the limited number
of clinical samples in this study, the sensitivity of the IL-4 assay
was equivalent to that of the IFN- test in that the same animals
gave positive responses in both tests (i.e., responses to PPD-M and
ESAT-6). The number of positive IL-4 responses in the naturally
infected animals was perhaps surprising, given the short sharp peak of
IL-4 responses observed for experimentally infected cattle, and
warrants further investigation.
Finally, M. bovis-specific IFN- and IL-4 responses were
compared with helminth-specific cytokine responses. Parasitic helminths are particularly potent inducers of IL-4 (18), and
experimental O. ochengi infection is known to induce both
IFN- and IL-4 responses in cattle (Graham et al., unpublished data).
The results suggest, first, a similar level of antigen-specific IFN-
production induced by infection with either M. bovis or
O. ochengi and, second, that the increased activity of
IL-4-producing cells in either infection following restimulation with
specific antigen in vitro was similar. However, there was a large
disparity in the spontaneous release of IL-4 by unstimulated PBMC in
these two very different infections of cattle, with high spontaneous
release in O. ochengi-infected animals compared to a
relatively low spontaneous release in M. bovis infected
animals, indicating a higher overall IL-4 activity in helminth infection.
In summary, this study describes the production of both IFN- and
IL-4 responses following the experimental infection of cattle with
M. bovis and shows the distinct response kinetics of these two cytokines. Cytokine response measurements in naturally infected M. bovis field reactors showed that assays based on the
detection of either IFN- or IL-4 responses were equally able to
differentiate infected from uninfected animals. Finally, an interesting
observation of positive IL-4 responses in low-dose M. bovis
challenge animals that remained SCITT suggests that the
measurement of IL-4 could provide additional information of disease
progression for use in investigations of the exposure to, and the
immunopathology of, bovine tuberculosis.
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ACKNOWLEDGMENTS |
|---|
We thank Adam Whelan (VLA) for providing the recombinant protein ESAT-6 and D. Gavier-Widen (VLA) for conducting the postmortem analysis of M. bovis experimentally infected animals. We also thank the IAH, Compton, Berkshire, United Kingdom, for providing the monoclonal antibodies IL-A58 and CC-304 and the recombinant bovine IL-4.
This work was funded by the United Kingdom Ministry of Agriculture, Fisheries and Food.
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FOOTNOTES |
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* Corresponding author. Mailing address: VLA, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 01932-357506. Fax: 01932-357684. E-mail: srhodes.vla{at}gtnet.gov.uk.
Editor: S. H. E. Kaufmann
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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