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Alternative activation of ruminant macrophages
by Fasciola hepatica
R.J. Flynn a, J.A. Irwin a, M. Olivier b, M. Sekiya a,J.P. Dalton c, G. Mulcahy a,*
a Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, College of Life Science,
University College Dublin, Belfield, Dublin 4, Irelandb Infectiologie Animale et Sante Publique, INRA, Centre de Tours, 37380 Nouzilly, France
c Institute of Biotechnology for Infectious Diseases, University of Technology Sydney, Sydney, NSW 2065, Australia
www.elsevier.com/locate/vetimm
Veterinary Immunology and Immunopathology 120 (2007) 31–40
Abstract
The helminth parasite, Fasciola hepatica, has a worldwide distribution and infects a wide variety of mammalian hosts, including
ruminants and man. In response to infection, these hosts mount a type 2 helper (Th2) response that is highly polarized and results in
the downregulation of type 1 helper (Th1) mechanisms. In a murine macrophage model F. hepatica induces alternative activation of
macrophages. These macrophages differ from classically activated cells in that they preferentially use arginase instead of inducible
nitric oxide synthase (iNOS) for metabolism of nitrogen. In this study we sought to characterize macrophage phenotype following
stimulation of the ovine cell line MOCL7 with recombinant F. hepatica enzymes and crude parasite extracts. An in vitro model using
the MOCL7 cell line was established and arginase levels in cells were used to determine the activation status of cells. Stimulation of
this cell-line in vitro with F. hepatica products induces alternative activation. We have also found a chitinase-like protein in
supernatants which is capable of differentiating alternatively activated from classically activated macrophages.
# 2007 Published by Elsevier B.V.
Keywords: Macrophage; Fasciola hepatica; Helminth; Immunoregulation; Alternative activation
1. Introduction
Infection with the helminth parasite Fasciola
hepatica (fasciolosis) causes major morbidity and
economic losses in sheep and cattle in temperate areas
(Spithill et al., 1999). Fasciolosis is also a major
zoonotic disease and it is estimated that 2.4 million
people worldwide are infected (Mas-Coma et al., 1999).
Problems with drug resistance in F. hepatica popula-
tions are also a growing concern (Coles, 2005). Progress
* Corresponding author. Tel.: +353 1 716 6180;
fax: +353 1 716 6185.
E-mail address: [email protected] (G. Mulcahy).
0165-2427/$ – see front matter # 2007 Published by Elsevier B.V.
doi:10.1016/j.vetimm.2007.07.003
in developing a vaccine against the parasite is ongoing
and appears the most promising option in combating the
disease in animal hosts (Dalton et al., 2003). Helminth
parasites, including F. hepatica, also cause concern due
to powerful immune regulatory abilities (Maizels et al.,
2004), that have the potential to decrease immune-
mediated protection against other diseases (Aitken
et al., 1978; Brady et al., 1999).
In ruminants F. hepatica can survive for long periods
of time, extending to a number of years in chronic cases
of the disease. Sheep, however, may succumb to acute
forms of the disease, where, if the worm burden is too
great, death may occur (Mulcahy et al., 1999). Both
hosts, however, show the same general patterns in terms
of immune response. Clery and Mulcahy (1998)
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–4032
reported that cattle showed reduced lymphocyte
responsiveness and IFN-g production after 3–4 weeks
of infection and also demonstrate a highly skewed
antibody response where IgG1 dominates (Clery et al.,
1996). Parasite persistence within the host suggests the
presence of immune regulatory networks. This reg-
ulatory and immune suppressive effect has been
examined in depth in the murine model of infection.
Mice infected with F. hepatica were seen to initiate Th2
effector mechanisms such as IL-4 and IL-5 secretion.
Th1 effector cytokines were suppressed when PBMC’s
from infected mice were re-stimulated in vitro with IFN-
g production being compromised. Antibody responses
were skewed towards IgG1, while IgG2a production
was diminished. The magnitude of these effects were
seen to be dependent on the final parasite burden
(O’Neill et al., 2000).
An understanding of how helminths are able to
generate immune environments which favour pathogen
persistence and chronic infection is important for
developing successful strategies for immunoprophy-
laxis. Modulation of macrophage and dendritic cell
function and interaction with toll-like receptors (TLR)
are thought to be some of the fundamental events
involved in establishing this immune regulation.
Macrophages function not only in the innate but also
in the adaptive immune response by presenting antigen
to primed T cells. The cytokine environment in which
they do so will influence the T cell subsets that later
develop. Macrophages metabolize L-arginine in two
ways either using inducible nitric oxide synthase
(iNOS) or arginase (arg). The differential regulation
of these enzymes is known to correspond to either
classical or alternative macrophage activation (Gordon,
2002). iNOS is used as a marker of classical activation,
while arginase is used to identify alternatively activated
macrophages (AAMF). It has reported that F. hepatica
infection induces AAMF in the murine model.
Furthermore, a recombinant version of a F. hepatica
enzyme peroxiredoxin (Prx) can also induce AAMF.
Inoculation of mice with Prx created a population of
macrophages with suppressed iNOS mRNA levels and
elevated TGF-b levels an immune regulatory cytokine
(Donnelly et al., 2005). The importance of the effects of
this molecule is two-fold. Prx is thought to act in the
defence of F. hepatica by detoxification of hydrogen
peroxide (H2O2) released by the host (Sekiya et al.,
2006). The level of H2O2 released by macrophages in
the presence of pathogens may also be determined by
the phenotype of macrophages. If F. hepatica has
evolved an enzyme to detoxify host H2O2 and modulate
the macrophage phenotype then it would appear that the
parasite defence mechanisms are tightly linked with
those of the host.
A number of other markers of alternative activation
are known to exist in the murine model, namely Ym1
and FIZZ1 which are non-functional members of the
chitinase family of proteins (Donnelly and Barnes,
2004). In the last decade, there have been several
chitinases and chitinase-like molecules identified in
higher mammals including chitotriosidase (Chit) and
acidic mammalian chitinase (AMCase) (Boot et al.,
2001; Renkema et al., 1995). AMCase, a 50 kDa
protein, is strongly expressed in the gut in the rodent,
where it has a pH optimum of 2 (Donnelly and Barnes,
2004). Unlike other members of this family of proteins,
AMCase also has enzymatic properties which involve
the 39 kDa N-terminal catalytic region which can
cleave chitin (Boot et al., 2001). Zhu et al. (2004) have
shown a possible role of AMCase expression in alveolar
macrophages to be under the control of IL-13, a Th2
cytokine, and also provide evidence for a role of this
protein in the pathology of asthma. Nair et al. (2005)
have also reported upregulated levels of chitinase and
FIZZ type proteins during nematode infection.
The purpose of this study was to evaluate the
activation of ruminant macrophages following exposure
to F. hepatica molecules by using arginase as a marker
of alternative activation. We also sought to identify if
chitinase or chitinase-like proteins could be used as
markers of macrophage activation in the ruminant
model.
2. Materials and methods
2.1. Cell line
The cell line used in these experiments was the ovine
monocyte cell line, MOCL7 (Olivier et al., 2001). This
cell line expresses membrane antigens representative of
blood derived monocytes, such as CD14 and poly-
morphic MHC II. The ability of MOCL7 cells to behave
similarly to native monocytes is also demonstrated by
phagocytosis of latex beads and a Salmonella abortu-
sovis strain (SAO Rv6).
2.2. Cell culture
MOCL7 cells were grown to confluency in 75 cm2
tissue culture flasks (Sarstedt) at 37 8C in 5% CO2,
using RPMI 1640 (Invitrogen), supplemented with
2 mM L-glutamine and 10% FCS (Sigma), containing
200 U/ml penicillin (Sigma) and 200 mg/ml strepto-
mycin (Sigma), 1 mM sodium pyruvate (Invitrogen),
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–40 33
5 � 10�5 mM mercapoethanol (Invitrogen), 1% non-
essential amino acids (Invitrogen). Cells were grown to
confluency and then collected by disassociating the cell
layers in Mg2+ and Ca2+ free trypsin/EDTA (Invitrogen)
for 10 min at 37 8C. Cells were collected in centrifuge
tubes and washed three times in warm Mg2+ and Ca2+
free Hanks Buffered Saline Solution, HBSS (Invitro-
gen). Following this, viable cells were counted using the
trypan blue method and resuspended in complete
medium at a final concentration of 1 � 106/ml.
Prior to stimulation, 500 ml of cell suspension was
added to every well of a 12 well flat-bottomed tissue
culture plate (Sarstedt). The cells were allowed to
adhere for 2–3 h and then the medium was removed.
Following this the wells were washed by rinsing in
Mg2+ and Ca2+ free HBSS and fresh medium was
added. Antigens were added at the indicated concen-
trations in final volumes of 20 ml. For control cultures,
the same volume of sterile PBS was added. Following
stimulation, the supernatant was removed and stored at
�20 8C until further use, and the cells were lysed using
1% Triton X-100 before freezing at �20 8C.
2.3. Antigen preparation
Liver fluke homogenate (LFH) and excretory-
secretory (ES) products were prepared as previously
described (Smith et al., 1993). Recombinant peroxir-
edoxin (Prx) was produced in an Escherichia coli
system also as described (Sekiya et al., 2006).
Recombinant peroxiredoxin (rPrx) was available in
our lab in two forms mutant (mu) and wildtype (wt).
rwtPrx behaves in a similar manner to the native forms
purified from ES (Sekiya, personal communication).
rwtPrx contains a 2 Cys active site, in the mutant form
the cysteine residue at position 47 has been mutated to a
tyrosine reside by a single nucleotide change.
2.4. Endotoxin removal
For preparation of endotoxin free F. hepatica
molecules polymyxin B columns (Pierce) or a phase
separation technique was used according to a previously
described method (Gao and Tsan, 2003a; Aida and Past,
1990). Briefly, columns were washed with 5 volumes of
1% sodium deoxycholate (Sigma) followed by 10
volumes of sterile PBS. rPrx or LFH was then added to
the column in aliquots of 250 ml and incubated for 1 h at
room temperature. They were eluted in sterile endotoxin
free PBS and following this the column was flushed
with sterile PBS. The phase separation method was also
used for endotoxin removal. Briefly proteins, adjusted
to 1 mg/ml in sterile endotoxin free PBS, were vortexed
with 5% Triton X-114, and incubated on ice for 5 min,
then at 37 8C for 5 min. Following the last incubation
solutions were centrifuged, in a microcentrifuge, at
5000 � g for 7 s at 37 8C. Following centrifugation, the
upper phase of the solution was collected, containing
the endotoxin free proteins. For all proteins used in
experiments, two cycles of phase separation were used.
After removal of endotoxin, molecules were either
given a prefix of t for polymyxin B column treated
molecules or pt for phase separated molecules. The
protein concentration was measured using the BCA
system (Pierce) with bovine serum albumin (BSA) as a
standard.
2.5. Endotoxin detection
The presence of endotoxin was detected in protein
solutions prior to and after treatments to remove
endotoxin using the Cambrex QCL-1000 Chromogenic
LAL Endpoint Assay. Briefly, 50 ml of sample was
incubated with 50 ml of LAL reagent for 10 min. 100 ml
of substrate solution was added and the reaction stopped
after 6 min with 100 ml of stop reagent (25% glacial
acetic acid). The absorbance was then measured at
405 nm. Endotoxin levels were quantified using a
standard curve and reported as EU/ml.
2.6. Nitric oxide measurement
Supernatants were tested in duplicate for nitric oxide
(NO) using the Griess reagent system (Promega).
Briefly, 50 ml of supernatant was added to wells of a 96
well plate (Sarstedt). To this 100 ml of sulfanimide
solution was added and the plate was incubated for
10 min in the dark at room temperature. Following this,
100 ml of N-1-napthylethylenediamine dihydrochloride
(NED) solution was added and the plate incubated
as before. Readings were taken at 570 nm and NO
concentration was determined by comparison with a
standard curve prepared from a 100 mM solution of
nitrate.
2.7. Arginase activity assay
Cell lysates were prepared by addition of 400 ml of
1% Triton X-100 (Sigma), and incubation on a rocking
platform for 40 min. Lysate (50 ml) was added to 50 ml
of Tris–HCl buffer, pH 7.5, and incubated at 55 8C for
10 min for enzyme activation. Following this, 25 ml of
the activated lysate was added to 25 ml of arginine
substrate at a concentration of 0.5 M (pH 9.7). This
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–4034
Fig. 1. Differential Regulation of iNOS and arginase in MOCL7 cells.
(a) Nitric oxide levels in collected supernatants were taken as indi-
cators of iNOS activity while arginase levels were measure in the same
cell lysates. These measurements were made after 48 h stimulation.
The nitric oxide levels of LPS stimulated cells were significantly
greater than those of PBS stimulated cells (P < 0.05). (b) The arginase
levels of LPS stimulated cells did not reach levels significantly greater
than the PBS control. The levels shown are the mean of three tested
cultures plus S.E.M.
mixture was incubated at 37 8C for 1 h. The reaction was
stopped by addition of 400 ml of acid stop solution,
comprising H2SO4 (96%), H3PO4 (85%), and H2O in a
ratio of 1:3:7. Colour was developed by adding 25 ml of
9% isonitrosopriopherone (Sigma) and heating to 103 8Cfor 45 min. A 1:20 dilution of beef liver homogenate was
used as a positive control. Two hundred microliters of
reaction mixture was added to wells of a 96 well plate and
the OD measured at 570 nm. The levels of urea in
samples were calculated by comparison with a standard
curve. One unit of enzyme activity was defined as the
quantity of enzyme that results in the formation of
1 mmol urea/min. On graphs arginase levels are given as
mU which is milli-units (mU) per 106 cells.
2.8. Chitinase assay
Chitinase activity was measured in supernatants as
previously described using a 96 well fluoregenic assay
(Boot et al., 2001). Briefly, 10 ml of supernatant was
mixed with 40 ml of McIlvaine buffer (100 mM citric
acid and 100 mM sodium phosphate) at the indicated
pH containing 0.25 mM of substrate. The substrate used
was 4-methylumbelliferyl b-D-N,N0-diacetylchitobiose
(4MU-chitobiose; Sigma–Aldrich). Following incuba-
tion of the sample with substrate/buffer for 2 h at
37 8C the reaction was stopped with 200 ml of 0.25 M
Gly/NaOH. Fluorescence was determined using a
Perkin-Elmer fluorimeter (excitation 365 nm; emissions
460 nm). Values are reported as nmol/h/ml following
reference to a standard curve generated from 4-
methylumbelliferone.
2.9. Statistical analysis
Data was tested for normality and then levels of
enzyme activity were tested for significant difference by
using the two-tailed Student’s t-test on MiniTab
software (Microsoft). Results were considered signifi-
cantly different if the P-value was <0.05.
3. Results
3.1. Differential production of arginase and
nitric oxide
To demonstrate that MOCL7 cells can regulate
arginase and iNOS in a similar manner to other MF
models we first stimulated cells with 5 and 10 mg/ml of
LPS from E. coli strain 111:B4 (Sigma–Aldrich). After
24 h arginase activity was detected in cells, however,
only trace amounts of NO were found. By 48 h after
stimulation, NO was detectable in LPS treated cells and
arginase levels were not significantly greater than PBS
treated cells (Fig. 1). At both 48 and 72 h NO was seen
to increase to levels significantly greater than those of
PBS treated cells (P < 0.05). It was noted that as NO
levels increased in LPS stimulated cells the arginase
levels decreased. This relationship indicates differential
regulation of arginase and iNOS in MOCL7 cells and
demonstrates their ability to undergo both alternative
and classical activation.
3.2. Alternative activation of macrophages by a
panel of F. hepatica molecules
A panel of F. hepatica derived molecules was next
used to stimulate the cells to determine their effect on
activation. The antigens used included rPrx, LFH, and
ES. After 48 h stimulation arginase activity was
detectable in all cells stimulated with F. hepatica
molecules. These levels were all significantly greater
than those found in PBS stimulated cells with Prx giving
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–40 35
Fig. 2. (a) Following 48 h stimulation with a panel of F. hepatica
derived molecules the arginase levels in cells lysates were measured.
The antigens LFH, ES, and rPrx all induced alternative activation. The
arginase levels differed as the dose of antigen increased, however, this
trend was not significant. (b) NO levels of the same cultures were
measured and only high doses of crude antigen resulted in minimal
NO production with levels never being above 1 mM. The results
represent the mean of three cultures plus S.E.M.
Fig. 3. A time course assay was conducted to determine when peak
arginase levels occur. (a) Cells stimulated with LFH were measured
for arginase levels 2, 6, 8, 24, and 48 h post-stimulation. From 2 h
arginase activity was seen in the cells, in this case activity in LFH
treated cells was significantly greater than controls. This trend con-
tinued until 24 h where activity peaked. Here activity in stimulated
cells was not only greater than PBS controls but also significantly
greater than the time point before or after (P < 0.05). (b) Cells were
stimulated with rmtPrx and arginase measured as above. Levels were
seen to rise over time. The peak of enzyme activity was seen to occur
at 24 h. Levels here are significantly greater than the time point before
and after. Levels of arginase induced by rwtPrx are significantly
elevated throughout the experiment when compared to the PBS.
Levels shown are the mean of three cultures.
the highest levels. A dose of LFH at 5 mg/ml produced
the lowest levels of arginase activity with 36 mU while
rwtPrx gave the highest levels of 74.4 mU. As seen in
Fig. 2a the level of arginase following stimulation was
dose dependent. Fig. 2b illustrates the lack of NO in the
supernatants from the same cultures. Only the highest
doses of LFH and ES, crude molecules induced NO
production. These levels failed to exceed 1 mM.
3.3. Maximal arginase levels
Having previously demonstrated that arginase
activity was detectable from 24 h post-stimulation
and that F. hepatica derived molecules can successfully
activate macrophages, we next sought to determine at
what point the peak response, in terms of arginase
activity, would occur. A time course study was
undertaken using LFH to stimulate cells for 2, 6, 8,
24, and 48 h. Following these periods of stimulation we
assayed cell lysates for arginase activity. From 2 h post-
stimulation, measurable arginase activity was present in
cells stimulated with LFH and PBS (Fig. 3a). These
levels were significantly greater than those found in
PBS treated cells with P values being 0.01 and 0.007,
respectively, for the two doses, 5 and 10 mg/ml. The
levels continued to increase until 24 h post-stimulation
where arginase activity was seen to peak at 69.8 and
77.1 mU for the 5 and 10 mg/ml doses, respectively. At
48 h, levels had dropped but remained elevated in
comparison to PBS controls, where levels were 36.1 and
41.9 mU, respectively, for the 5 and 10 mg/ml doses.
Fig. 3b shows a similar pattern for rwtPrx, peak levels of
arginase were found at 24 h where the activity obtained
here was significantly greater than the activity seen at 8
and 48 h.
3.4. Peroxiredoxin activates host macrophages
Time course experiments showed that arginase levels
peak again at 24 h post-stimulation with rwtPrx, with
the two doses 5 and 10 mg/ml producing 66.5 and
61.3 mU, respectively. To rule out the possibility that
the enzyme activity of rwtPrx was necessary for this
response, we tested the two forms (mu and wt)
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–4036
Fig. 4. Two forms of rPrx were tested to ensure the enzyme active site
of Prx was not responsible for activation of cells. Macrophages were
treated with 5 mg/ml of rwtPrx or rmuPrx and arginase activity in cell
lysates was determined after 24 h. At this point both treatments had
activated the cells, but no significant difference was seen between the
two treatments. The levels are the mean of three cultures plus SEM.
Table 1
Endotoxin levels of F. hepatica molecules
Endotoxin level (EU/ml) treatmenta
Molecule Native Polymyxin B Phase separation
LFH 0.613 0.066 NDb
Prx 0.103 0.018 ND
a Molecules were tested in the LAL assay either in a native
(untreated) form, or following purification over a polymyxin B
column, or following phase separation.b ND—not detected, endotoxin could not be detected in the samples
tested.
Fig. 5. Removal of endotoxin, by two methods, does not inhibit
alternative activation. (a) LFH purified over a polmyxin B column,
tLFH, produces levels of arginase similar to untreated controls. Cells
were treated with 10 mg/ml of LFH or tLFH. (b) Cells treated with
5 mg/ml of untreated or phase separated molecules behave in the same
manner in terms of arginase production. The levels are the mean of
three cultures plus S.E.M.
simultaneously at a dose of 5 mg/ml. Fig. 4 shows that
arginase levels were similar in cultures given both
forms. Both forms again induced levels of activity
significantly greater than PBS controls, with rwtPrx
inducing 66.5 mU of enzymatic activity while rmuPrx
gave 73.9 mU. The levels produced by rwtPrx and
rmuPrx treatmenent were not significantly different
from each other (P > 0.05).
3.5. Removal of endotoxic residues from F. hepatica
molecules fails to stop alternative activation of
macrophages
Endotoxic residues are ubiquitous and may be
responsible for the activation of macrophages in many
settings (Gao and Tsan, 2003a,b). The possibility that
the activating effect seen in response to stimulation with
LFH or Prx could be a result of endotoxic contamination
had to be ruled out. LFH is a crude antigen prepared
from whole fluke extracts and Prx is produced in a
recombinant E. coli system, where there is a strong
possibility of contaminating LPS being present. To
examine the effect endotoxins have on activation of
macrophages by F. hepatica molecules we removed
endotoxins over a polymyxin B column (t) or by phase
separation (pt). The levels of endotoxin detected prior to
and following treatment are shown in Table 1.
Cells were stimulated as before for 24 h with LFH
and tLFH at a dose of 10 mg/ml. LFH induced 77.1 mU
of arginase activity while tLFH induced 77.4 mU of
activity. These levels are significantly greater than those
found in the PBS controls for the experiment, indicating
that both antigens had alternatively activated the
macrophages. Also, analysis of the individual levels
of activity indicated that there was no significant
difference between the levels induced by LFH or tLFH
(P > 0.05) (Fig. 5a). For protein purified by phase
separation we treated cells for 24 h with 5 mg/ml of
LFH and Prx, and their respective phase separated
forms, ptLFH and ptPrx. No significant changes in
arginase levels were detected between the native form
or phase treated form of the molecule. LFH produced
22.7 mU of arginase activity while ptLFH gave
21.22 mU of enzyme activity (P > 0.05) (Fig. 5b).
When the cells were treated with Prx, the arginase
activities were found to be 20.94 and 19.55 mU
(P > 0.05) for Prx and ptPrx, respectively. This clearly
indicated that the response of cells to F. hepatica
molecules was not due to any contaminating endotoxic
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–40 37
Table 2
Effect of TCA on enzyme activity
% TCA 0.05 1 10 15
% reduction in activity 3.14 2.09 72 95
Prx generated supernatants were incubated for 30 min at room tem-
perature with an equal volume of the indicated TCA solution. Samples
were then tested under standard conditions using McIlvaine buffer at
pH 4.5, the enzyme activities were then compared to a control sample,
incubated with water, and the percentage differences in activity was
calculated.
residues but to an intrinsic activating ability of these
molecules.
3.6. Chitinase activity as a marker of alternative
activation in ruminant hosts
To determine whether chitinase could be used as a
marker for activation in ruminant macrophages we
assayed the supernatants of stimulated cells after 48 h.
We found that chitinase activity was indeed present and
that there were higher levels present in the supernatants
taken from cells stimulated with LFH, as compared to
LPS or PBS stimulated cells (data not shown). To
determine the optimum conditions under which this
chitinase was active we assayed three types of super-
natants. Supernatants were taken from cells stimulated
with LFH and rPrx at 10 ug/ml and LFH at 5 ug/ml.
Peak activity occurred between pH 3 and 4 (Fig. 6a).
The peak pH was further pinpointed to 2.5 (Fig. 6b).
This indicated that the chitinase present in the super-
natant was likely to be AMCase since chitriosidase
activity is maximal at a range of pH 4.5–5.2 (Renkema
Fig. 6. Optimum pH of the chitinase enzyme found in supernatants
was determined by buffering McIlvaine buffer to the indicated pH. (a)
Three types of supernatant were used, these were generated by
stimulating macrophages with LFH at 5 or 10 mg/ml and Prx at
10 mg/ml. The chitinase activity in all three supernatants peaked
between pH 2 and pH4. To further pinpoint the pH a range of 1.6–
3.7 was assayed using the same Prx generated supernatant as before. It
can be seen that the pH which produced the optimal activity was 2.5,
indicating an acidic chitinase enzyme.
et al., 1995) and at pH 5, chitinase activity in our
samples had halved as compared to that at pH 2.5. We
further characterized the enzyme by addition of TCA to
the supernatant samples prior to being assayed. Table 2
shows that up to 1% TCA solution had little effect on the
activity of the enzyme, while incubation with TCA
solutions of 10 and 15% reduced enzyme activity by 72
and 95%, respectively. As AMCase is acid stable and
chitriosidase is not, this suggests that the enzyme found
here is similar AMCase (Boot et al., 2001).
To investigate the possibility of using chitinase
activity as a marker of macrophage activation we
assayed supernatants taken from cells stimulated with
either LPS or rwtPrx for 48 h. Following this time
supernatants were collected. Assay of the samples
indicated that a difference in chitinase activities was
present. The LPS dose of 1 mg/ml produced higher
levels of chitinase activity compared to a dose of 5 mg/
ml with chitinase activity being 12.93 and 6.97 nmol/h/
ml, respectively. rwtPrx stimulated cells produced
chitinase activites of 27.42 and 26.45 nmol/h/ml for
cells given a dose of 5 and 10 mg/ml, respectively. In
comparison to the LPS 1 mg/ml generated chitinase
levels, they were significantly greater for the 5 and
10 mg/ml dose where P = 0.0003 and 0.003, respec-
tively (Fig. 7a). In comparison, NO was detected only in
LPS stimulated cells when compared to controls or Prx
activated cells (Fig. 7b). The contrast in NO and
chitinase levels indicates that chitinase activity could
serve as a marker of alternative activation.
4. Discussion
Our results to date demonstrate that molecules
derived from F. hepatica are capable of alternatively
activating macrophages in vitro. This is in agreement
with previous findings (Donnelly et al., 2005). Our
results add to the data available on macrophage
activation by helminths. To date AAMF have been
shown to be induced in models of infection using
Schistosoma mansoni (Herbert et al., 2004), Trichnella
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–4038
Fig. 7. (a) Supernatants taken from activated cells were tested for
chitinase activity 48 h after stimulation. Both LPS treated cultures
failed to induce chitinase activity above that of the control, while Prx
treated cultures had levels of chitinase significantly greater than either
the control or LPS treated cultures (P < 0.05). (b) Levels of NO were
also measured, NO was not detected in cells treated with PBS or both
doses of Prx. However, NO production was detected in cells treated
with increasing doses of LPS. The levels are the mean of three cultures
plus SEM.
spiralis (Dzik et al., 2004), and Taenia crassiceps
(Terrazas et al., 2005). Our results are the first to
describe the response of ruminant-derived macrophages
to F. hepatica molecules. AAMF may prove to be an
important step in the establishment of the immune
regulatory environment during F. hepatica infection.
These types of immune responses are important because
of their inhibitory effect on Th1 responses in F. hepatica
infected animals and the associated hyporesponsiveness
of associated immune mechanisms. Our results also
highlight the ability of proteins secreted by parasites to
influence the immune environment. Since the Prx active
site is not responsible for the activation of cells; the
mechanism of action remains to be defined.
Classically activated macrophages (CAMF) are
usually associated with Th1 responses and thereby
combat bacterial infections, via free radical production.
AAMF are linked to Th2 responses, which occur during
asthma, fungal and parasitic infections. The differential
regulation of these two macrophage phenotypes has been
shown to correlate with the Th1/Th2 cytokine balance
and T cell subsets (Hesse et al., 2001; Munder et al.,
1998). The modulatory capacity of helminth-induced
macrophages in murine models is well documented.
Using a T. crassiceps model of infection, Terrazas et al.
(2005) have shown that T cell anergy induced by AAMF
was contact dependent, while simultaneously being
independent of IL-10, IFN-g, and NO. Examination of
the surface of these macrophages revealed that pro-
grammed death ligand 1 (PD-L1) and PD-L2 were
upregulated. Neutralization of PD-L1, PD-L2, or their
receptor PD-1 restored lymphocyte responsiveness.
Smith et al. (2004) found S. mansoni infection to have
the same effect on MF where typical Th2 cytokines IL-4
and IL-13 and regulatory cytokines IL-10 and TGF-b
were found to have no role in the MF dependent cell
anergy. Blockade of PD-L1 restored lymphocyte
responses. This evidence extends the dual role MF play
in immunity, as APCs they may have a critical role in
induction of downstream responses.
Ym1 and Fizz-1 are commonly used markers of
AAMF in the murine model, they are also non-
functional members of the chitinase family of proteins.
Nair et al. (2005) found these proteins to be upregulated
in nematode infection. This study also showed an
increase in AMCase expression; however, this expres-
sion was restricted to the lungs. The role of chitinases in
helminth infections is still unclear; however, since Ym1
and Fizz are non-functional it remains to be defined
whether they may act as chemokines. Webb et al. (2001)
found Ym2, whose expression was IL-13 dependent, to
have weak chemotactic ability. However, the chitinase
identified in our work is fully functional and appears
similar in enzymatic properties to AMCase. A role for
AMCase has been identified in the murine model of
asthma and its presence was detected in human
bronchoalveolar (BAL) samples taken from asthmatics
(Zhu et al., 2004). In a murine model of a sensitized
airway both mRNA and protein levels of AMCase were
upregulated in an IL-13 dependent manner. Further
examination showed that AMCase was incapable of
upregulating cytokine expression on its own. Using in
situ hybridization and immunohistochemistry AMCase
was localized to macrophages and epithelial cells
derived from the airway. Chitinases, and in particular
AMCase, have also been implicated in the asthmatic
response and have been identified as potential
therapeutic targets (Donnelly and Barnes, 2004; Elias
et al., 2005). A chitinase like protein, SI-CLP, has also
been linked to AAMF (Kzhyshkowska et al., 2005). In
this study AAMF were generated by stimulating
cells with IL-4 and levels of SI-CLP were examined
and found to be upregulated. It was found that
human cartilage glycoprotein 39 (HC-gp39), a chitinase
family member, inhibited cellular responses to IL-1 and
R.J. Flynn et al. / Veterinary Immunology and Immunopathology 120 (2007) 31–40 39
TNF-alpha (Ling and Recklies, 2004). This would mirror
the action of cytokines produced by AAMF. The
potential role that chitinases have in helminth infections,
either as functional enzymes or possible chemokines,
must be investigated further.
To conclude, the results presented here demonstrate
the induction of alternative activation of a ruminant
derived MF cell line by F. hepatica molecules. We have
also identified a novel ovine chitinase that appears
similar to AMCase and is associated with the induction
of AAMF in this model. The role of AMCase warrants
particular study since it is a functional enzyme and
could also have a possible chemokine role in promoting
eosinophilia, which is an important process in helminth
infections. The role of AAMF need to be further
investigated in respect of their effect on bacterial
coinfections in F. hepatica hosts needs be determined
and their role in the establishment of adaptive immune
responses in chronic Th2 responses must also be fully
examined.
Acknowledgements
R.J. Flynn would like to thank The Irish Research
Council for Science Engineering and Technology for
funding under the EMBARK Initiative. Funding for
the study was also provided by the EU Commission
under Framework 6, Project ref.FOOD-CT-2005-
02305-DELIVER. The authors would also like to thank
Drs. Donnelly and O’Neill for helpful discussions.
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