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Veterinary Immunology and Immunopathology 114 (2006) 135–148

Cytokine and antibody subclass responses in the intestinal lymph

of sheep during repeated experimental infections with the

nematode parasite Trichostrongylus colubriformis

Anton Pernthaner *, Sally-Ann Cole, Lilian Morrison, Richard Green,Richard J. Shaw, Wayne R. Hein

AgResearch Limited, Wallaceville Animal Research Centre, PO Box 40063, Ward Street, Upper Hutt, New Zealand

Received 22 November 2005; received in revised form 30 June 2006; accepted 3 August 2006

Abstract

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-a and IFN-g genes, and parasite-specific IgM, IgG1, IgG2, IgA

and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite

Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep

consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-g did

not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and naı̈ve S

sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge.

Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous

exposure to nematodes. Genes for TNF-a and IFN-g were also transiently up-regulated, but to a lesser extent and more typically

after primary challenge. Naı̈ve sheep of both genotypes produced relatively little antibody response after primary challenge. A

second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for

adult antigen-specific IgA and larval antigen-specific IgG1. In naı̈ve S line sheep, the larval-specific IgA and IgG2 response

appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels

prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode

infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-g gene

expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total

IgE in lymph plasma.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Intestinal immune response; Afferent lymph; Efferent lymph; Cytokines; Antibody subclass; Nematode infection; Sheep

Abbreviations: AIL, afferent intestinal lymph; AILC, afferent

intestinal lymph cells; EIL, efferent intestinal lymph; EILC, efferent

intestinal lymph cells; FEC, faecal egg count; DC, dendritic cells

* Corresponding author. Tel.: +64 4 529 0577; fax: +64 4 529 0380.

E-mail address: tony.pernthaner@agresearch.co.nz

(A. Pernthaner).

0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2006.08.004

1. Introduction

The acquisition of immunity to gastro-intestinal

nematodes has seemingly paradoxical features. While

on the one hand, the immune mechanisms acting against

different parasites are uniquely adapted to specific life-

cycle stages (Meeusen et al., 2005), on the other hand

the host immune system appears to mount a stereotypic

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148136

Th2 response which activates a set of generic effector

mechanisms that protects against most intestinal

nematode parasites (Finkelman et al., 2004). The

inability to understand better the complex mix of

regulatory mechanisms which underlie immunity and

susceptibility to gastro-intestinal nematodes accounts,

at least partly, for the relative lack of success in

developing nematode vaccines.

Immune responses against gastro-intestinal nema-

todes are mounted in what is arguably the most

complex immunological micro-environment in the

body. The intestinal immune system mediates crucial

and often opposing immunological functions such as

protective responses to intestinal pathogens and

tolerance to harmless commensal bacteria and dietary

proteins in order to maintain homeostasis of the gut

(Mowat, 2003). To achieve these outcomes, the gut

immune system contains several structural and

functional specialisations for the uptake and proces-

sing of antigens and the dissemination of immune

responses. Achieving a better understanding of the

events unfolding within this regional microenviron-

ment during nematode infections will be critical to

the development of new vaccines. Research into the

role of the lymphatic system recently gained wide

spread attention as crucial functions of the normal

immune system and its functions in human diseases

are being recognized (Skobe et al., 2001; Swartz and

Skobe, 2001). A major cell population trafficking in

lymph is highly mobile dendritic cells (DC) which

form sentinels in the periphery, and their migrating

through afferent lymphatic vessels is crucial for the

execution of their functions (Randolph et al., 2005).

In large domestic animal species cannulation of the

afferent and efferent lymph ducts of a lymph node has

greatly facilitated the study of the immune system

functions, both under physiological conditions and

during immune responses to various pathogens. Many

of these studies utilize the lymphatic system draining

the skin of sheep (Gohin, 1997). The lymphatic

system draining from the wall of the small intestine,

which is an important site of gut-associated lymphoid

tissue (Lowden and Heath, 1993), has been less

explored.

Recently, we reported methods for long-term

collection of afferent and efferent lymph draining the

small intestine of sheep and showed that this approach

could be used to study the regional immune response to

diverse pathogens such as Mycobacterium avium

subsp. paratuberculosis or the nematode parasite

Trichostrongylus colubriformis (Hein et al., 2004).

We subsequently used this approach to compare the

nature of the regional response during a single

experimental infection with T. colubriformis in sheep

with different levels of genetically determined resis-

tance. When compared to susceptible-line (S) sheep,

resistant-line (R) sheep expressed higher levels of some

Th2 cytokines in immune cells migrating in intestinal

lymph (Pernthaner et al., 2005a), and developed

enhanced total- and nematode-specific IgE responses

in their intestinal lymph plasma (Pernthaner et al.,

2005b).

In the work reported here, we extended these

studies in two ways. Firstly, regional immune

responses were monitored in animals during two,

three, or four successive nematode infection cycles,

each of 21-day duration. This approach enabled the

detection of immunological events that require

repeated infections, as does the development of

protective immunity. Secondly, in addition to mon-

itoring the expression levels of the six cytokine genes

measured during primary infection in the earlier study,

we also measured nematode-specific IgA, IgG1, IgG2

and IgM in intestinal lymph at daily intervals

throughout the infection. The combination of cytokine

gene expression and antibody sub-class levels in

intestinal lymph provides a significantly more detailed

description of regional immune response parameters

during nematode infection than has been achieved

previously.

2. Material and methods

2.1. Surgical procedures

All animal experiments and surgical procedures

were approved by the Animal Ethics Committee of the

Wallaceville Animal Research Centre. Efferent lym-

phatic vessels draining from lymph nodes in the region

of the upper or mid jejunum were cannulated using a

vinyl cannula. A two-stage surgical procedure was used

to gain access to afferent lymph draining from the small

intestine as described previously (Pernthaner et al.,

2002). Briefly, in the first stage, mesenteric lymph nodes

which drain from the jejunum were removed, and in a

second stage operation, a reconstituted ‘pseudo-

afferent’ lymphatic vessel was located and cannulated

enabling continuous sampling of lymph. Methods for

long-term collection and characterization of afferent

and efferent lymph draining from the small intestine of

sheep were reported in detail elsewhere and are repeated

here only in brief to enable understanding of the overall

procedures and outline of the experiment (Hein et al.,

2004).

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 137

2.2. Animals and experimental design

The aim of this study was to detect differences in the

cytokine gene expression and antibody subclass

responses in afferent and efferent intestinal lymph

between nematode resistant (R) and susceptible (S) line

sheep during two experimental infections with T.

colubriformis. Cytokine genes that are important in

mediating nematode disease or nematode immunity and

that are indicative for either a Th1 (IFN-g) or a Th2 type

response (IL-4, IL-5, IL-13), or are regulative (IL-10) or

predominantly pro-inflammatory (TNF-a) were

assayed.

Sheep were derived from the Wallaceville R (n = 6)

or nematode S (n = 5) sheep lines which differ at least

36-fold in their autumn faecal egg counts under natural

challenge on pasture (Bisset et al., 2001; Morris et al.,

2000). A total of 11 male sheep, aged between one and

two years was included in the study. Animals were

challenged orally on day 0 with 50,000 infective larvae

of T. colubriformis and the infection was terminated at

day 21 by drenching with oxfendazole (5 mg/kg body

weight; Systamex1, COOPERS). Sheep were then re-

challenged 7 days after drenching. All sheep had a

patent cannula for more than 20 days after the second T.

colubriformis infection. In two separate field-primed R-

line sheep, the cannulae remained patent throughout

three or four 21-day cycles of challenge infection

respectively, allowing extended observations in these

animals.

Animals were either nematode naı̈ve (pen-raised;

Rn, n = 3; Sn, n = 2) or had experienced previous

natural polygeneric nematode challenge (field-primed;

Rp, n = 3; Sp, n = 3). These animals were fitted with

either afferent (n = 5) or efferent (n = 6) lymphatic

cannulae.

2.3. Parasitology

Infective T. colubriformis larvae were cultured under

standard procedures from eggs obtained from faeces of

a mono-specifically infected sheep. Faecal egg counts

were performed at weekly intervals throughout the

duration of the experiment using the modified

McMaster method (Whitlock, 1948).

2.4. Sampling

Five to 15 ml of fresh lymph was collected daily over

a period of 30–60 min into sample tubes under aseptic

conditions to allow sampling of cells and lymph plasma.

Cells were separated from lymph by centrifugation,

washed once in PBS and re-suspended in RNAlater

(Ambion) to preserve the integrity of mRNA. Cell and

lymph samples were stored at�20 8C until subjected to

either RNA extraction and reverse transcription or

antibody subclass analysis.

2.5. RNA extraction and reverse transcription

RNA extraction and reverse transcription was

performed as described previously (Pernthaner et al.,

2005a). Briefly, RNAlater was removed prior to RNA

extraction by centrifugation and total RNA isolated

from cells using TRI REAGENT1LS (Molecular

Research Center, Inc.) based protocol according to

the manufacturer’s instructions. Total RNA yield was

calculated based on the absorption at 260/280 nm. First-

strand cDNA synthesis was performed using Super-

ScriptTMII RNAse H- reverse transcriptase (InVitrogen)

and poly(A) oligo(dT)12–18 primer to reverse tran-

scribe up to 5 mg of total RNA according to the

manufacturer’s protocol, and adjusted to an initial

concentration of 20 ng/ml RNA. cDNA was stored at

�20 8C until used for real time PCR.

2.6. Primer design, real time PCR and

quantification of gene expression

For each target gene a primer pair was selected using

the Primer Express software (Applied Biosystems) for

use in a SYBR1Green real time PCR assay, which were

described previously (Hein et al., 2004).

Cytokine gene expression was detected by real time

PCR using a GeneAmpTM 5700 sequence detection

system in combination with sequence detector software

(Applied Biosystems, Foster City, CA, USA) as

described previously (Pernthaner et al., 2005a). Briefly,

reactions were assembled in duplicates in optical 96-

well reaction plates using SYBR1Green PCR master

mix (Applied Biosystems). For each sample a melting

curve was generated after completion of amplification,

and analysed in comparison to the positive and negative

controls. Mean threshold cycle (CT) values of duplicate

samples were used for analysis.

The comparative CT method was employed for

relative quantification where the amount of target is

normalized to an endogenous reference (housekeeping

gene) (User Bulletin #2: ABI PRISM 7700 Sequence

Detection System; Applied Biosystems). The CT

indicates the fractional cycle number at which the

amount of amplified target reaches a fixed threshold,

and DCT represents the difference in threshold cycles

for the target and housekeeping gene.

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148138

Fig. 1. FEC in individual naı̈ve (n) and field-primed (p) R and S line

sheep during two cycles of experimental nematode infection. Animals

were infected on days 0 and 28 with 50,000 L3 T. colubriformis and

drenched 21 days after each challenge. FEC were determined 21 days

after each infection. FEC in Rp sheep were significantly (P < 0.05)

lower than in Rn, Sp and Sn sheep after the first challenge infection.

Both Rn and Rp sheep had significantly (P < 0.01) lower FEC than Sn

and Sp sheep after the second challenge infection. Numbers in

parentheses indicate the challenge infection.

2.7. Enzyme immunoassay (EIA) to detect specific

antibody subclass responses in lymph

Levels of T. colubriformis specific IgA, IgM, IgG1 and

IgG2 antibody subclasses in lymph were determined

using a non-competitive antibody sandwich immunoas-

say (EIA) as described previously (Douch et al., 1994).

Briefly, microtitre plates were coated either with T.

colubriformis excretory secretory products (TcESL3)

obtained from infective larvae or with somatic antigen

obtained from adult worms (TcAd) and incubated with

diluted lymph or serum samples. For IgG1 the samples

were diluted 1:400, for IgA, IgG2 and IgM 1:10.

Nematode specific antibody subclasses were then

detected using subclass-specific anti-ovine mAb and a

peroxidase based detection system. Lymphatic IgE levels

were detected using a non-competitive antibody sand-

wich EIA as described previously (Pernthaner et al.,

2005b; Shaw et al., 1997).

2.8. Nematode larvae and antigen preparation

Infective larvae (L3) of T. colubriformis were

obtained from cultures of faeces taken from a mono-

specifically infected sheep. T. colubriformis excretory

secretory product was obtained from exsheathed

infective larvae as described previously (Douch et al.,

1994). Somatic antigen was prepared by homogenizing

adult worms under liquid nitrogen. Soluble protein was

extracted in 5 mM Tris buffer pH 7.6 and stored at

�70 8C until used for assay.

2.9. Statistical analysis

Cytokine gene expression data were analysed on DCT

values using procedures for pulse analysis and REML

analysis of group means (Patterson and Thompson, 1971)

as described previously (Pernthaner et al., 2005a) with

the exception that subgroups were pooled when

appropriate. Pulse analysis for individual cytokine gene

expression profiles was done using the variables control

chart method, which is used for tracking processes over

time and is able to detect special cases not fitting into the

baseline variation, as described previously (Pernthaner

et al., 2005a). Data for antibody subclasses were analysed

using the same procedures as for cytokine gene

expression. FEC data were analysed as an unbalanced

two-way ANOVA after log(x + 100) transformation. The

non-parametric Kruskal–Wallis test was used to compare

time points of when antibody levels started to rise above

background between naı̈ve R and S line sheep. P < 0.05

was considered to be significant.

3. Results

3.1. Parasitology

FEC determined 3 weeks after the first challenge

infection showed that the Rp animals had significantly

(P < 0.05) lower FEC than the Rn, Sp or Sn sheep. At

this stage FEC of the Rn tended to be lower than for the

Sn sheep but this difference did not achieve signifi-

cance. The difference in FEC between R and S sheep

diverged further after the second challenge and both the

Rn and Rp groups showed significantly (P < 0.01)

lower FEC than the corresponding S groups (Fig. 1). As

expected FEC determined at weeks 1 and 2 after each

challenge infection were always 0.

3.2. Comparison of primary and secondary

nematode challenged cytokine responses in

intestinal lymph cells

3.2.1. IL-13 gene expression in lymph cells during

primary and secondary challenge infection of naı̈ve

and field-primed R and S sheep

The genotype of the animal but not the type of lymph

had a significant influence on the IL-13 gene expression.

Therefore, data for AILC and EILC were combined

for analysis. Nematode naı̈ve (n = 3) and field-primed

R sheep (n = 3) expressed significantly higher mean

levels of the IL-13 gene than S line sheep during two

experimental infections with T. colubriformis

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 139

Fig. 2. Cytokine gene expression in AILC and EILC of individual naı̈ve (n) and field-primed (p) R and S sheep. Animals were infected on days 0 and

28 with 50,000 L3 T. colubriformis and drenched 21 days after each challenge. FEC determined at days 21 and 49 revealed large differences in egg

production between R and S sheep (for details see Fig. 1). IL-13 was significantly (P < 0.01) higher expressed in the R than S sheep. The time

required for the nematode development (L3, third stage larvae; L4, fourth stage larvae; EAd, early adult nematodes) is indicated. Results are

presented as DCT values, which is the difference in threshold cycles for the target and housekeeping gene. Therefore, low DCT values represent high

gene expression and high DCT values represent low gene expression. (a) Within naı̈ve R and S sheep the mean IL-13 gene expression did not differ

significantly between the first and second challenge infection. Large but transient increases occurred in the R sheep 8–14 days after primary and 2–8

days after secondary challenge. (b) For field-primed R and S sheep large but transient increases in the IL-13 gene expression occurred 1–7 days after

primary and secondary challenge. Only R sheep had an additional period of IL-13 gene up-regulation 15–21 days after primary challenge.

(P < 0.01; data from individual naı̈ve and field-primed

R and S sheep are shown in Fig. 2a and b). Naı̈ve R

sheep were able to respond to primary experimental

infection with a trend for IL-13 up-regulation, which

continued during the secondary infection, but differ-

ences of mean expression between cycle one and two

were not statistically significant (Table 1). The mean IL-

13 gene expression in the naı̈ve S sheep (n = 2) during

two challenge infections was low resulting in an

increase from an 11-fold to a 32-fold difference from

the first to the second challenge between R and S sheep.

In contrast, the 14-fold difference in the mean IL-13

gene expression seen between field-primed R (n = 3)

and S sheep (n = 3) during the first challenge declined to

a six-fold difference during the second challenge due to

a tendency for down-regulation in the R animals. In two

field-primed R sheep the mean expression of the IL-13

gene remained elevated throughout a third and fourth

challenge (data not shown).

When different developmental stages of T. colu-

briformis were taken into account, a large transient

over-expression of the IL-13 gene was consistently seen

in naı̈ve R animals (n = 3) approximately 8–14 days

post primary challenge, at the time when mostly L4

larval stages are present (Fig. 2a). In these sheep,

termination of the infection at day 21 resulted in a

decline of the IL-13 gene expression to approximately

pre-challenge levels within 7 days. When naı̈ve R sheep

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148140

Table 1

Predicted means at day 10 from the first and second cycle (21 days each) of experimental infection with T. colubriformis

Animals Cycle 1 Cycle 2 P diff

Mean S.E. (mean) Mean S.E. (mean)

IL-13 Rn 3 9.52 1.63 7.58 1.63

IL-13 Sn 2 12.84 1.88 13.15 1.88

IL-13 Rp 3 7.29 1.19 8.79 1.19

IL-13 Sp 3 11.08 1.19 11.89 1.19

IL-5 n 5 17.10 1.08 16.54 1.08

IL-5 p 6 13.27 0.31 15.84 0.31 **

IFNg n 5 8.12 0.74 8.88 0.74

IFNg p 6 7.67 1.47 9.83 1.47

IL-4 n 5 11.03 0.44 10.75 0.44

IL-4 p 6 10.05 0.74 11.24 0.74

IL-10 Rn 3 11.16 0.28 10.74 0.28

IL-10 Sn 2 10.88 0.33 12.19 0.33 **

IL-10 Rp 3 8.80 0.69 11.08 0.69 *

IL-10 Sp 3 9.73 0.69 11.14 0.69

TNFa AILC n 2 6.74 0.85 6.98 0.85

TNFa EILC n 3 10.00 0.74 8.46 0.74

TNFa AILC p 3 7.93 0.44 7.22 0.44

TNFa EILC p 3 6.26 0.44 8.05 0.44 **

Gene expression of IL-5 and IL-10 was significantly lower in the indicated subgroups during the second cycle of infection. Results are presented as

mean of DCT values, which is the difference in threshold cycles for the target and housekeeping gene. Therefore, low DCT values represent high

gene expression and high DCT values represent low gene expression.* P < 0.05.

** P < 0.01.

where subjected to a second infection a large transient

over-expression of the IL-13 gene appeared approxi-

mately 2–8 days after challenge, at the time when L3

and early L4 larval stages are present (Fig. 2a). A

transient up-regulation of the IL-13 gene expression

was also found in the corresponding S sheep (n = 2)

after primary and secondary challenge infection but no

consistent pattern was seen.

Nematode challenge of field-primed R (n = 3) and

S (n = 3) sheep resulted in a transient up-regulation of

the IL-13 gene approximately 1–7 days after primary

infection (with the exception of one S sheep). In the R

sheep, a second period of IL-13 up-regulation

followed approximately 15–21 days after challenge,

which was not seen in S sheep (Fig. 2b). Within 7

days after drenching IL-13 gene expression levels

returned approximately to pre-challenge levels. For

both the R and S sheep a second challenge resulted in

a large transient IL-13 gene up-regulation approxi-

mately for 1–7 days after infection, and thereafter no

consistent trends were observed. These taken together

indicate that in field-primed sheep antigens produced

by infective larvae are strong inducers of the IL-13

gene.

3.2.2. IL-4, IL-5, IL-10, TNF-a and IFN-g gene

expression in response to primary and secondary

nematode infection of naı̈ve and field-primed sheep

Within each cycle of challenge the mean gene

expression values for each of IL-4, IL-5, IL-10, TNF-a

and IFN-g did not differ significantly between R and S

sheep and therefore data were pooled for analysis.

Similarly, data for AILC and EILC were also pooled

where appropriate.

In naı̈ve sheep (n = 5) mean gene expression levels

for IL-4, IL-5, IL-10, TNF-a and IFN-g did not change

significantly between the first and second challenge,

with the exception of a significant down-regulation of

the IL-10 gene expression in the Sn sheep (n = 2;

Table 1).

Field-primed sheep (n = 6) had elevated levels of

mean IL-5 and IL-10 gene expression during the first

challenge, which were 16- and 4-fold higher respec-

tively, than for naı̈ve sheep. Mean IL-5 gene expression

in field-primed sheep (n = 6) and mean IL-10 expres-

sion in Rp (n = 3) sheep were significantly lower during

the second when compared to the first challenge

(Table 1). The mean gene expression for IL-4, TNF-a

and IFN-g was steady during two cycles of infection

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 141

with the exception of the down-regulation of TNF-a in

EILC of field-primed sheep (n = 3; P < 0.01; Table 1).

The gene expression of IL-5, TNF-a and IFN-g was

modulated in both AILC and EILC in response to

primary and secondary challenge infection. Large

transient increases in IL-5 gene expression levels were

seen in individual naı̈ve sheep during the first and

second challenge (data for individual R and S sheep are

given in Fig. 3a). Consistent trends were only seen for

IL-5 in R (n = 3) sheep which showed a transient

increase approximately 7–10 days after primary

challenge and 1–9 days after secondary challenge,

though base line expression levels differed substantially

between animals (Fig. 3a). Large transient increases in

Fig. 3. IL-5 gene expression in AILC and EILC of individual naı̈ve (n) or fie

with 50,000 L3 T. colubriformis and drenched 21 days after each challenge

production between R and S sheep (for details see Fig. 1). The time required

larvae; EAd, early adult nematodes) is indicated. Results are presented as DC

housekeeping gene. Therefore, low DCT values represent high gene expressi

gene expression did not differ significantly between the first and second chall

occurred in particular 7–10 days after the first and 1–9 days after the second

higher during the first than second challenge infection of field-primed (p) R an

after each challenge infection.

IL-5 gene expression were also seen in field-primed R

and S sheep (with the exception of an R animal during

the second challenge) in particular approximately 1–7

days after the first and second challenge (data for

individual R and S sheep are given in Fig. 3b). In field-

primed animals the main peaks for IL-5 gene expression

appear to coincide with the major peaks in IL-13

expression approximately 3–7 days after challenge.

The expression of the TNF-a gene was also

transiently up-regulated in particular after primary

challenge but no consistent trends within sheep lines

were seen (data not shown). Large but transient

increases in the IFN-g gene expression occurred in

naı̈ve and field-primed R and S sheep in particular

ld-primed (p) R and S sheep. Animals were infected on days 0 and 28

. FEC determined at days 21 and 49 revealed large differences in egg

for the nematode development (L3, third stage larvae; L4, fourth stage

T values, which is the difference in threshold cycles for the target and

on and high DCT values represent low gene expression. (a) Mean IL-5

enge infection of naı̈ve (n) R and S sheep. Large but transient increases

challenge infection. (b) Mean IL-5 gene expression was significantly

d S sheep. Large but transient increases occurred in particular 1–7 days

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148142

during the first challenge but animal-to-animal varia-

tions were large and no consistent trends within line

animals were seen (data not shown). In contrast,

changes in the IL-4 and IL-10 gene expression within

cycles of challenge infections were comparatively small

(data not shown).

3.3. Antibody responses in afferent and efferent

intestinal lymph

3.3.1. Lymphatic antibody subclass response of

naı̈ve R and S line sheep

No significant genotype related difference for the

lymphatic IgG1, IgM and IgE antibody subclass

responses was found in naı̈ve and field-primed animals

during two challenge infections. Therefore, data from R

and S animals were pooled for analysis. The only

genotype related differences were significantly higher

adult antigen-specific IgA and larval antigen-specific

IgG2 levels in the Rn when compared to the Sn sheep

(data not shown). Rn (n = 3) but not Sn sheep (n = 2)

had a significant increase in larval antigen-specific IgG2

levels between the first and second challenge infection

(Table 2).

Table 2

Predicted means of antibody subclass levels at day 10 from the first and secon

Animals Cycle 1

Mean S.E. (m

L3IgA n 5 �2.190 0.164

L3IgA p 6 �0.707 0.351

AdIgA n 5 �2.052 0.160

AdIgA p 6 �1.126 0.332

L3IgG1 n 5 �2.025 0.135

L3IgG1 p 6 �0.056 0.376

AdIgG1 n 5 �2.079 0.407

AdIgG1 p 6 �1.724 0.251

L3IgG2 Rn 3 �2.047 0.117

L3IgG2 Sn 2 �1.814 0.135

L3IgG2 Rp 3 �1.367 0.313

L3IgG2 Sp 3 �2.076 0.313

AdIgG2 n 5 �2.453 0.183

AdIgG2 p 6 �1.970 0.259

L3IgM n 5 �1.775 0.217

L3IgM p 6 �1.492 0.140

AdIgM n 5 �2.474 0.202

AdIgM p 6 �1.859 0.118

IgE n 5 0.879 0.358

IgE p 6 2.384 0.133

Levels of adult antigen-specific IgA (AdIgA), larval antigen-specific Ig

significantly from the first to the second cycle of infection in naı̈ve (n) s

which did not increase further during challenges. Data for IgA, IgG1, IgG2 a

IgE are mean values of U/ml.

Overall, antibody subclass responses of naı̈ve R and

S line sheep to primary experimental infection were

small, but for some animals adult antigen-specific IgA

and IgG1 and total IgE was detected, particularly at the

later stage of the challenge (data for IgA and IgG1 in

AIL and EIL from individual animals are given in

Fig. 4a and c, data for IgE not shown). A second

nematode challenge resulted in an increase in the

lymphatic IgA, IgG1, IgG2 and IgE production (data for

larval and adult antigen-specific IgA and IgG1 in AIL

and EIL from individual animals are given in Fig. 4a–d;

data for IgG2 and IgE not shown). This increase

achieved statistical significance for the adult antigen-

specific IgA (P < 0.01) and larval antigen-specific IgG1

(P < 0.05) responses (Table 2). Little larval but no adult

antigen-specific IgM response was seen during primary

infection which did not change during the secondary

challenge of naı̈ve sheep (Table 2). R line sheep (n = 3)

tended to respond earlier than S line sheep (n = 2) with a

TcES IgA and IgG2 response, although these differ-

ences did not achieve significance ( p = 0.08) due to the

small group size.

These findings show that naı̈ve R and S line sheep

were able to respond at the late stage of a primary

d cycle (21 days each) of experimental infection with T. colubriformis

Cycle 2 P diff

ean) Mean S.E. (mean)

�1.770 0.164

�0.653 0.351

�1.310 0.160 **

�0.958 0.332

�1.542 0.135 *

�0.023 0.376

�1.438 0.407

�1.712 0.251

�1.455 0.117 **

�1.923 0.135

�1.433 0.313

�1.995 0.313

�2.194 0.183

�1.890 0.259

�1.819 0.217

�1.558 0.140

�2.472 0.202

�1.849 0.118

1.539 0.358

2.654 0.133

G1 (L3IgG1) and larval antigen-specific IgG2 (L3IgG2) increased

heep. Field-primed (p) sheep had existing high levels of antibodies

nd IgM represent mean values of natural log transformed OD; data for

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 143

Fig. 4. IgA and IgG1 subclass responses in AILC and EILC of individual naı̈ve (n) R and S sheep. Animals were infected on days 0 and 28 with

50,000 L3 T. colubriformis and drenched 21 days after each challenge. FEC determined at days 21 and 49 revealed large differences in egg

production between the R and S sheep after the second challenge infection (for details see Fig. 1). The time required for the nematode development

(L3, third stage larvae; L4, fourth stage larvae; EAd, early adult nematodes) is indicated. (a) Adult antigen-specific IgA levels during the first and

second nematode challenge of individual naı̈ve sheep (n = 5). Mean adult antigen-specific IgA levels increased significantly from the first to the

second nematode challenge (P < 0.01). (b) Larval (L3) antigen-specific IgA levels during the first and second nematode challenge of individual

naı̈ve sheep (n = 5). (c) Adult antigen-specific IgG1 levels during the first and second nematode challenge of individual naı̈ve sheep (n = 5). (d)

Larval (L3) antigen-specific IgG1 levels during the first and second nematode challenge of individual naı̈ve sheep (n = 5). Mean L3 antigen-specific

IgG1 levels increased significantly from the first to the second nematode challenge (P < 0.01).

experimental nematode infection with secretion of adult

antigen-specific lymphatic IgG1. With some exceptions

these animals were also able to respond to the second

nematode challenge with a nematode specific IgA,

IgG1, IgG2, and total IgE production. The antigen-

specific IgA and IgG2 response appeared to be

enhanced in R when compared to S line sheep.

3.3.2. Lymphatic antibody subclass response of

field-primed R and S line sheep

Field-primed R (n = 3) and S line sheep (n = 3) had

established larval and adult antigen-specific lymphatic

IgA, IgG1, IgG2, IgM and total IgE levels prior to

challenge, and mean values did not differ significantly

between the first and second challenge infection

(Table 2). IgG1, IgG2 and IgM levels remained

approximately steady throughout two challenge infec-

tions (data not shown). In two field-primed R sheep

these already elevated antibody subclass levels did not

change significantly during a third or fourth challenge

infection (data not shown).

In contrast to IgG1, IgG2 and IgM responses, large

transient increases in lymphatic IgA and total IgE levels

were seen after primary and secondary experimental T.

colubriformis infection in both line sheep (data for EIL

and AIL from individual R and S sheep are shown in

Fig. 5a–c). These transient increases appeared to be

biphasic with the first peak appearing approximately 2–

13 days and the second 16–20 days after the first

challenge. When developmental stages are considered

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148144

Fig. 5. IgA and IgE subclass responses in AILC and EILC of individual field-primed (p) R and S sheep. Animals were infected on days 0 and 28 with

50,000 L3 T. colubriformis and drenched 21 days after each challenge. FEC determined at days 21 and 49 revealed large differences in egg

production between the R and S sheep after each challenge infection (for details see Fig. 1). The time required for the nematode development (L3,

third stage larvae; L4, fourth stage larvae; EAd, early adult nematodes) is indicated. (a) Adult antigen-specific IgA levels during the first and second

nematode challenge of individual field-primed sheep (n = 6). (b) Larval (L3) antigen-specific IgA levels during the first and second nematode

challenge of individual field-primed sheep (n = 6). (c) Total IgE levels during the first and second nematode challenge of individual field-primed

sheep (n = 6).

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 145

the first increase coincides with L3 and early L4 stages,

and the later peak with adult nematode parasites.

Transient increases were also seen during the second

challenge, again approximately 2–14 days after

challenge, but thereafter no consistent trends were

observed. Although these increases tended to be higher

in R than S line sheep, no significant difference was

detected due to the large animal-to-animal variation.

These findings demonstrate that field-primed R and S

line sheep were able to respond in a similar manner to

two experimental nematode infections in addition to

total IgE with antigen-specific IgG1, IgA, IgG2 and

IgM production and the response was directed against

both larval excretory/secretory and adult somatic

nematode antigens.

4. Discussion

Nematode challenge of the host is met by a range of

humoral and cellular responses but immunity is usually

Th2 dependent. Recent evidence suggests that the Th2

pathway is not a ‘default pathway’ but one that is

actively instructed by mechanisms that are only

beginning to be understood. Areas of intensive

investigation include studies on the role of antigen

presenting cells in Th2 response development, the

inhibitory function of IL-10, regulatory T-cells and

decoy receptors on chronic Th2-mediated inflamma-

tion, and the role of chitinases in mediating Th2 disease

which accelerated our understanding of the contribution

of these factors to Th2 immunity (Ramalingam et al.,

2005).

Recently, we described and validated reliable

methods for long-term collection of ovine afferent

and efferent intestinal lymph using experimental

infection with T. colubriformis as a model for Th2-

type and natural infection with Mycobacterium avium

spp. paratuberculosis as Th1-type responses (Hein

et al., 2004). In consecutive studies on Th2-type

responses we used the same lymphatic cannulation

procedures in the Wallaceville R and S line sheep while

they were subjected to a single experimental infection

with T. colubriformis. This approach allowed us to

monitor, without any need for further in vitro

stimulation, the in vivo expression levels of several

cytokine genes in cells migrating in lymph directly from

those sites where anti-nematode immune responses are

generated without compromising the integrity of the

immune system. We found that R line sheep were able

to respond to a single experimental nematode infection

in the presence of IFN-g with a significant up-regulation

of the genes for IL-13, IL-5 and TNF-a when compared

to S-line sheep (Pernthaner et al., 2005a). IgE levels

were also found to be higher in intestinal lymph from R

line sheep than S line sheep (Pernthaner et al., 2005b).

This study extends the previous findings and

demonstrates that cannulation of intestinal afferent

and efferent lymphatic vessels can also effectively be

used to investigate over periods of two or more

challenge infections in vivo intestinal nematode

immune responses. We were able to characterize

cytokine gene expression profiles and antibody subclass

responses during this prolonged period of time which

enabled us to compare primary with secondary

challenge responses.

The animals used in this study exhibited the expected

phenotypic differences (Bisset et al., 2001; Morris et al.,

2000) and overall, FEC were lower in the R than S

groups. The immune response to the second challenge

of Rn and to both challenge infections of Rp sheep was

fully protective, but S sheep remained susceptible to

nematode infection throughout the course of the

experiment. FEC were also lower in Rn than Sn sheep

following primary infection, although this difference

did not achieve statistical significance.

The genotype related difference in the mean IL-13

gene expression, which was observed previously during

primary infection (Pernthaner et al., 2005a), was

maintained during the second challenge of both naı̈ve

and field-primed sheep. In the case of naı̈ve animals, the

mean IL-13 gene expression increased in R, but not S

sheep between the first and second challenge, although

this increase in the R sheep did not achieve statistical

significance. The only genotype related change between

the first and second challenge was seen in the mean IL-

10 gene expression with a significant down-regulation

in the Sn and Rp animals during the second challenge.

In humans, enhanced secretion of IL-10 by CD14+

monocytes is associated with reduced chlamydia

antigen-specific lymphocyte proliferative and IFN-g

responses (Ohman et al., 2006). However, in this study

the down-regulation in IL-10 did not coincide with an

increase in the IFN-g response nor did it significantly

enhance the expression of the Th2 effector cytokines IL-

5 and IL-13 (Maizels, 2005). The reasons for the

genotype dependent IL-10 effects remain unknown and

require further study.

Compartment related differences in mean gene

expression between the first and second challenge

infection were generally small and only the down-

regulation of TNF-a in EILC of field-primed animals

was significant. Maybe some of the key functions of

TNF-a such as promoting activation of innate cells and

release of pro-inflammatory cytokines or influencing

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148146

the transition from innate to adaptive immunity

(Rahman and McFadden, 2006), are not required to

the same extent in animals previously exposed to

natural nematode infections. Interestingly, this down-

regulation is only seen in cells migrating in efferent

lymph, a cell population consisting almost exclusively

of lymphocytes. In contrast, afferent lymph cells, which

contain other cell subsets with the potential of TNF-a

expression like antigen presenting cells and natural

killer cells, retained the elevated TNF-a gene expres-

sion throughout two cycles of challenge. This indicates

that TNF-a mediated activation of antigen presenting

cells and natural killer cells, and differentiation of T and

B cells, remains steady at a mucosal level during

nematode challenge.

The only other significant difference in mean gene

expression levels between the first and second challenge

was the down-regulation of the IL-5 gene during the

second challenge of field-primed sheep. These animals

had elevated mean IL-5 gene expression levels during

the first challenge, which induced IgE production and

may have mediated nematode immunity via induction

of eosinophilia and mastocytosis. These functions of IL-

5 are well established in both sheep (Bao et al., 1996;

Gill et al., 2000) and rodents (Zhou et al., 1996) and

maintenance of these functions, once initiated, may not

require a permanent IL-5 stimulus.

Interestingly, in all experimental groups, mean IL-4

and IFN-g gene expression levels remained constant

over two challenge infections indicating that in sheep,

the transcription of IL-4 and IFN-g genes is not

modulated to the same extent as in laboratory animals

following nematode exposure (Deschoolmeester and

Else, 2002; Finkelman et al., 1997).

Although the overall changes in mean gene

expression levels between the first and second challenge

were comparatively small, large transient increases of

IL-13, IL-5, TNF-a and IFN-g over shorter periods of

time were present in many animals. When we attempted

to relate these temporary changes with the biology of

the nematode infection, stage-specific induction, in

particular of IL-13 and IL-5, became obvious. These

stage-specific transient increases, however, were not

entirely consistent between cycles of challenge infec-

tion, genotype or pre-challenge status. For the naı̈ve R

but not S sheep the induction of the IL-13 gene in

response to primary infection appears to be induced by

L4 larvae. When these R sheep were re-challenged, a

transient up-regulation appeared earlier in the course of

the infection indicating induction by L3 larvae.

Transient IL-13 up-regulation generally of lower

magnitude occurred also in the naı̈ve S sheep but these

peaks could not be related to any developmental stages.

In contrast, when field-primed R and S sheep were

subjected to a primary challenge, antigens produced by

L3 larvae appeared to induce an initial up-regulation of

the IL-13 gene, but a second peak at a later stage of the

infection was only seen in R sheep, when L4 and adult

nematodes would have been present. When field-

primed sheep were re-challenged, S sheep showed a

large transient up-regulation of the IL-13 gene at the

early stage of the infection.

Our data further show that in addition to IL-13, gene

expression of IL-5, IFN-g and TNF-a was clearly

modulated during two nematode challenge infections.

Nematode challenge of naı̈ve R sheep resulted in a

transient up-regulation of the IL-5 gene. Although

small animal numbers and a large animal-to-animal

variation might have masked some of the underlying

trends, a consistent up-regulation of the IL-5 gene was

induced at a time when L4 larval stages were present.

On the other hand, each of the two challenge infections

in the majority of field-primed R and S sheep resulted in

a transient up-regulation of the IL-5 gene expression

levels which tended to coincide with peaks in IL-13

gene expression. The major peaks for IL-13 and IL-5

gene expression appeared approximately 3–10 days

after challenge, which was consistent for field-primed

R and S sheep during both challenge infections and for

naı̈ve R sheep during the first challenge. These taken

together provide evidence that nematode antigens

expressed at specific developmental stages are power-

ful inducers of IL-13 and IL-5 gene transcription.

Furthermore, antigens produced by infective T.

colubriformis larvae are considered to be effective

inducers of Th2-type cytokines which is well known for

filarial infections (Osborne and Devaney, 1998) or

glycans derived from several nematode species (Tawill

et al., 2004). The identification of such T. colubriformis

nematode antigens, especially of the IL-13 inducing

antigens, is considered as having great potential in the

discovery of vaccine candidates, as the critical role of

IL-13 in mediating nematode immunity is generally

well recognized (Finkelman et al., 2004; Grencis,

1997; Grencis, 2001; McKenzie et al., 1998; Wynn,

2003). We found previously that IL-5 was over-

expressed in AILC of R when compared to S sheep

(Pernthaner et al., 2005a), but mice are able to develop

and maintain immunity to nematode infection in the

absence of IL-5 (Dent et al., 1999; Fallon et al., 2002;

Hokibara et al., 1997). This makes the discovery of

predominately IL-5 inducing nematode antigens a less

attractive option for identifying potential vaccine

candidates.

A. Pernthaner et al. / Veterinary Immunology and Immunopathology 114 (2006) 135–148 147

Primary challenge of naı̈ve and field-primed sheep

resulted in repeated transient up-regulation of the genes

for TNF-a and IFN-g in most animals, which could not

be consistently related to any developmental stages.

This transient over-expression was less prominent and

somehow controlled during the second challenge. As

seen previously for primary challenge (Pernthaner et al.,

2005a), field-primed R line sheep were able to maintain

a protective immune response during the second

challenge infection in the presence of and despite

transient up-regulation of the IFN-g gene.

To our knowledge this is the first extensive report on

lymphatic antibody subclass titres in response to

repeated nematode infections. However, as mentioned

before, the group size of subgroups was small and

possible effects of lymphatic compartment or genotype

might not have been detected. Nonetheless, our data

show some interesting findings. Mean antibody titres for

antigen-specific IgG1, IgG2, IgA and total IgE

increased from the first to the second challenge of

naı̈ve sheep and this increase was statistically sig-

nificant for adult antigen-specific IgA and for larval

antigen-specific IgG1. Increases in other antigen-

specific subclass responses might not have been

identified as such as some naı̈ve animals were able to

respond to primary infection with a specific antibody

production. The only genotype related difference

detected was the significantly higher increase in larval

antigen-specific IgG2 response between the first and the

second challenge of Rn but not Sn sheep. Field-primed

sheep had previously experienced natural nematode

challenge and had existing lymphatic antibody titres

prior to challenge. As expected, these animals were able

to respond to nematode challenge with an early up-

regulation of lymphatic antibody subclass titres but

mean values did not increase significantly following a

second challenge. The pattern of the overall lymphatic

antibody responses was similar to those observed

previously in serum (Douch et al., 1994) with IgG1 and

IgG2 tending to continuously increase over time until

reaching a plateau.

In contrast to IgG1 and IgG2, the increase in antigen-

specific IgA and total IgE following primary and

secondary challenge of field-primed sheep appeared to

be biphasic, with a first peak coinciding with the

presence of infective and L4 larvae, and a second peak

by which time adult stages would have developed. This

is a strong indication of the stage-specific induction of

IgA and IgE, especially when the short serum half-life

of IgA and IgE is taken into consideration (Klobasa and

Werhahn, 1989; Curtis and Bourne, 1973; Vieira and

Rajewsky, 1988).

Although compromised by the small group size, our

data also provide evidence that R line sheep are able to

respond earlier than S line animals with a larval antigen-

specific IgA and IgG2 response. The response to adult

antigens was similar for both lines. Evidently, in R line

sheep, antigens produced by L3 and L4 larval

developmental stages serve as effective inducers of

multi-valent anti-nematode immune responses marked

by the expression of Th2-type cytokines and IgA and

IgG2. It remains to be elucidated if S line sheep respond

with a dominating IL-13 and IL-5 response in

conjunction with a larval antigen-specific IgA and

IgG2 production similar to that one seen in R line sheep

once protective nematode immunity is developed.

Taken together these results demonstrate that during

repeated nematode infections, the intestinal micro-

environment of sheep is transiently skewed towards Th2

cytokine dominance, although IFN-g gene expression

continues. The up-regulation of Th2 cytokines appears

to be biphasic with induction by antigens predominantly

produced by early larval developmental stages and, with

some restrictions, by adult nematodes. This response is

accompanied by increases of nematode-specific IgG1,

IgA, IgG2 and IgM, as well as of total IgE in lymph

plasma.

Acknowledgements

This work was supported by the Foundation for

Research, Science & Technology. We thank Roger

Littlejohn for advice about the statistical analyses used.

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