Download Ixodes scapularis the Saliva of the Lyme Disease Vector Tick

Identification of an IL-2 Binding Protein in
the Saliva of the Lyme Disease Vector Tick,
Ixodes scapularis
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of June 14, 2017.
R. Dean Gillespie, Marc C. Dolan, Joseph Piesman and
Richard G. Titus
J Immunol 2001; 166:4319-4326; ;
doi: 10.4049/jimmunol.166.7.4319
http://www.jimmunol.org/content/166/7/4319
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Copyright © 2001 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Identification of an IL-2 Binding Protein in the Saliva of the
Lyme Disease Vector Tick, Ixodes scapularis1
R. Dean Gillespie,2* Marc C. Dolan,† Joseph Piesman,† and Richard G. Titus*
I
nteractions between blood-feeding arthropods and their hosts
may have profound implications for the transmission of vector-borne pathogens (1– 4). Of all the blood-feeding vector
arthropods, hard ticks, by virtue of their protracted feeding period,
represent an extreme example of evasion of their host’s hemostatic
defenses and immune response (5– 8). Ticks are obligate bloodfeeding ectoparasites, with many species remaining attached for
periods of 3–15 days (9, 10). Many tick species are important
vectors of disease worldwide. Ixodes scapularis is the main North
American vector of Lyme disease, now the most prevalent vectorborne disease reported in the United States (11), as well as human
granulocytic ehrlichiosis and babesiosis (12–15). Because ticks deliver the molecules used to confound host defenses in their saliva,
this secretion is an important feeding and vector competence factor
for these ectoparasites (9).
Several pharmacological activities have been characterized in
tick saliva with important roles in blood feeding, including vasodilators, anticoagulants, and anesthetics (5, 6, 8, 9, 16). This body
of observations makes it clear that ticks have evolved a remarkable
array of biochemical weapons to keep the host at bay and assure
their own success as a species. The protective functions of vector
saliva may also have the important, though presumably inadvertent, effect of potentiating the transmission of various pathogens to
hosts through the alteration of the immunologic microenvironment
*Department of Pathology, College of Veterinary Medicine and Biomedical Sciences,
Colorado State University, Fort Collins, CO 80523; and †United States Department of
Health and Human Services, Public Health Service, Centers for Disease Control and
Prevention, National Center for Infectious Diseases, Division of Vector-Borne Infectious Diseases, Fort Collins, CO 80522
Received for publication April 7, 2000. Accepted for publication January 19, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant AI27511 from National Institutes of Health (to
R.G.T.).
2
Address correspondence and reprint requests to Dr. R. Dean Gillespie, Department
of Pathology, Colorado State University, Fort Collins, CO 80523. E-mail address:
[email protected]
Copyright © 2001 by The American Association of Immunologists
at the feeding site (1, 2) or the lymph nodes draining the site of
attachment (17–20).
Many examples of immunomodulatory functions of tick saliva
have been described in various tick species that are disease vectors
to humans and livestock (reviewed in Ref. 7 and 21). Much attention has been given to the nature of the immune response to ticks
as well as to the effects of tick infestations on the host’s immune
competence (7, 9, 21). Tick infestation of mice has been shown to
result in significant modulations of cytokine production, with a
general pattern of the inhibition of proinflammatory and Th1 cytokines accompanied by enhancement of Th2 cytokines (19, 22, 23).
It has been observed that the saliva or salivary gland extracts
(SGE)3 of several tick species, including I. scapularis (16, 24),
profoundly inhibit the proliferative response of mitogen-stimulated
T lymphocytes. This phenomenon has been demonstrated with
mouse T cells by using Con A (16, 23–28), PHA (24), and antiCD3 (20). This effect has also been demonstrated on bovine lymphocytes with Con A stimulation (29, 30). This putative immunomodulatory phenomenon is particularly intriguing because of the
central role played by T cells in the orchestration of the acquired
immune response (31). Determining the mechanism of this inhibitory effect of saliva may have important ramifications regarding
the inability of the host to mount effective immune responses to
ticks and the pathogens they transmit.
Previous work with I. scapularis saliva has shown that this phenomenon is mediated by a protein(s) due to its lability to trypsin
treatment. This inhibitory effect was not dependent on mouse haplotype or the T cell mitogenic lectin used to drive proliferation
(24). It was also shown that the high levels of PGE2 in tick saliva
cannot account for the degree of inhibition of mouse T cell proliferation caused by saliva (24). In contrast, evidence that PGE2 in
Boophilus microplus saliva is the mediator of this effect on bovine
lymphocytes does exist (29). Furthermore, it was demonstrated
3
Abbreviations used in this paper: SGE, salivary gland extracts; IL-2BP, IL-2 binding
protein; TIP, T cell inhibitory protein; SBTI, soybean trypsin inhibitor; SC, spleen
cells; rm, recombinant mouse; rh, recombinant human; bio, biotinylated; IGBP, Ig
binding proteins; DTH, delayed-type hypersensitivity.
0022-1767/01/$02.00
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A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme
disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells
with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a
mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by
using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination
of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a
protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any
parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell
proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick
T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human
T cells and CTLL-2 cells grown in the presence of human IL-2. The Journal of Immunology, 2001, 166: 4319 – 4327.
4320
IDENTIFICATION OF AN IL-2 BINDING PROTEIN IN TICK SALIVA
with ELISA and bioassays that treatment of spleen cells (SC) with
tick saliva resulted in a decrease in IL-2 production (24). This
observation has also been made by others working with different
tick species (20, 23, 27, 30).
Our present work was undertaken to determine the mechanism
by which tick saliva nonspecifically inhibits T cell proliferation.
We report here on the identification of a proteinaceous tick salivary factor that binds murine and human IL-2. Furthermore, we
demonstrate that tick saliva inhibits mitogen-driven human T cell
proliferation.
Materials and Methods
Animals
Adult C57BL/6 mice, ⬎6 wk old, were obtained from National Cancer
Institute (Bethesda, MD). Female New Zealand White rabbits, 5–7 wk old,
were obtained from Western Oregon Rabbit (Philomath, OR).
Human PBMC preparations
Reagents and chemicals
The anti-CD3 mAb-producing clone 145-2C11 (CRL-1975) (33) was obtained from American Type Culture Collection (Manassas, VA). [3H]TdR
(5 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Pilocarpine (P-6503), PHA-L (leucoagglutinin; L-2769), trypsin (T-2271), and
soybean trypsin inhibitor (SBTI; T-6522) were obtained from Sigma (St.
Louis, MO). Recombinant mouse (rm)IL-2 (212-12), recombinant human
(rh)IL-2 (200-02), and rmIFN-␥ (315-05) were obtained from PeproTech
(Rocky Hill, NJ).
Ticks and saliva collection
All experiments were done with saliva from field-isolated ticks. Unfed
adult female ticks were collected by flagging vegetation at a site in Bridgeport, CT (34) during the fall (September-November) in 1996 –1999. For
saliva collection, ticks were allowed to feed on the ears of rabbits for 5–7
days. Near-replete and replete ticks were removed and immobilized, a
finely drawn capillary tube was fitted over their mouthparts, and 2–5 ␮l of
5% pilocarpine in methanol was applied topically to their dorsa (35). Saliva
was collected over 1–2 h in a 37°C environment, pooled, and stored at
⫺70°C until used. For cellular assays, saliva was sterilized with a 0.22-␮m
syringe filter before use. Except when indicated, the results depicted in the
figures are from independent batches of saliva; however, each experiment
described has been replicated with different batches of saliva.
Proliferation assays
Mouse SC were prepared as described previously at 5 ⫻ 106 cells/ml in
supplemented DMEM (24) and seeded at 5 ⫻ 105 well. Cultures were
maintained at 37°C, 5% CO2 during the assays. The cells were stimulated
with an anti-CD3 mAb culture supernatant at 1:500 in the presence or
absence of saliva (in triplicate) as described in Fig. 6. After 24 h, the
cultures were pulsed with 1 ␮Ci/well of [3H]TdR for 18 h followed by
harvesting and scintillation counting to determine the proliferative
response.
Human PBMC were plated at 1 ⫻ 106 cells/ml in 200 ␮l of medium (see
above for preparation details). Saliva was added to triplicate wells as indicated in Results and Fig. 1 and immediately stimulated with 2 ␮g/ml
PHA. The cultures were maintained at 37°C, 5% CO2 during the assay; at
24 h, the wells were pulsed with 1 ␮Ci/well [3H]TdR for 18 h before
harvesting and scintillation counting.
CTLL-2 cell assays
CTLL-2 cells were maintained in complete RPMI 10 medium in the presence of 1 U/ml human IL-2 (36). All assays were performed 2–3 days after
the last cell feeding. Cells were washed three times in sterile PBS and
reseeded at 5000 cells/well in a final volume of 50 ␮l in the presence of
human or mouse IL-2 and saliva as described in the Results and Fig. 7. The
IL-2 and IFN-␥ ELISAs
The IL-2-specific and IFN-␥-specific ELISAs were performed using
matched sets of rat mAbs obtained from BD PharMingen (San Diego, CA),
according to the manufacturer’s suggested protocol, on Nunc F96 Maxisorp plates (439454; Nunc, Naperville, IL). A rat anti-mouse IL-2 capture
mAb (rat IgG2a; 18161D) was used to capture IL-2. Biotinylated rat antimouse IL-2 mAb (rat IgG2b; 18172D) was used to detect captured IL-2. A
rat anti-mouse IFN-␥ mAb (rat IgG1; 18181D) was used to capture IFN-␥,
and the biotinylated rat anti-mouse IFN-␥ mAb (rat IgG1; 18112D) was
used to detect captured IFN-␥. Both assays were developed with avidinHRP (A-3151; Sigma), and 3,3⬘,5,5⬘-tetramethylbenzidine (TMB; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used as the substrate.
Preincubation of saliva with mouse IL-2 and IFN-␥ was done in 96-well
polypropylene clusters (Costar 3790; Corning Glass, Corning, NY). A serial dilution of saliva was prepared in DMEM supplemented with 5% FCS.
A serial dilution of the cytokine mouse IL-2 or IFN-␥ was also prepared in
DMEM supplemented with 5% FCS. Equal volumes of each serially diluted reagent were combined on transverse axes, 0 –1 ␮l saliva on one axis
and 0 – 4 pg of cytokine on the other, and incubated for 2– 4 h at 37°C. The
saliva-cytokine mixtures were then transferred to the capture Ab-coated
plate and incubated overnight at 4°C. The ELISA was completed using
standard procedures.
A control ELISA designed to test for possible Ab-binding by plated
saliva was performed as follows. Saliva (1 ␮l/well) was immobilized in
wells as described below. Following a wash step, wells were blocked with
Superblock (37535; Pierce, Rockford, IL). After another wash step, each of
the Abs used in the IFN-␥ and IL-2 ELISAs was added to the wells at the
concentrations used in the ELISAs and incubated for 2 h at room temperature. The samples were washed again and then incubated with a 1:500
dilution of HRP-conjugated goat anti-rat IgG (H⫹L) (14-16[hypehn]12;
Kirkegaard & Perry Laboratories) in 5% FCS/1⫻ PBS (pH 7.4) for 45 min
before a final wash and developed as described above.
Biotinylation of cytokines
rmIL-2, rhIL-2, and rmIFN-␥ were biotinylated using aminohexanoyl-biotin-N-hydroxysuccinimide ester (Zymed, San Francisco, CA) and standard protocols for biotinylation of proteins in solution (38). To clear the
biotinylated cytokines of excess quenched aminohexanoyl-biotin-N-hydroxysuccinimide ester and exchange buffers, the biotinylation reactions
were filtered on Microcon ultrafiltration units with a molecular mass cutoff
of 10 kDa that were passivated with Tween 20 following the manufacturer’s suggested procedure (Millipore, Bedford, MA). The filter was washed
twice with 400 ␮l 1⫻ PBS plus MgCl2 and CaCl2, and then the biotinylated cytokine was collected from the membrane in 100 ␮l 1⫻ PBS plus
MgCl2 and CaCl2.
The concentration of the recovered biotinylated cytokines was determined using competitive capture ELISAs with unlabeled cytokines of
known concentration as standards (39). The concentration of the cytokines
before dilution were: biotinylated (bio)-rmIL-2, 1.26 ng/␮l; bio-rhIL-2, 0.3
ng/␮l; and bio-rmIFN-␥, 1.34 ng/␮l.
Assays with plate-bound tick saliva
A serial dilution of tick saliva was plated on Nunc F96 Maxisorp plates in
standard ELISA carbonate buffer (pH 9.6) overnight at 4°C. After washing
with 1⫻ PBS (pH 7.4), the plates were blocked with 3% BSA/0.1% Tween
20/1⫻ PBS (pH 7.4). Following another wash step, an excess of biormIL-2, -rhIL-2, or -rmIFN-␥ was added to the plate for 4 h at room
temperature; the plate was then washed again. A blank control of salivacoated wells processed without a biotinylated cytokine incubation step was
also run. The plate was then incubated with avidin-HRP for 30 min followed by a final wash step and development with tetramethylbenzidine
reagents (Kirkegaard & Perry Laboratories). The reactions were stopped
with sulfuric acid before the absorbance was read at 450 nm. Before graphing, the blank control absorbance values were subtracted from the data, and
the absorbance values were normalized to account for differential levels of
biotinylation of the various cytokines used. The specific activity of each
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Blood was obtained from healthy human donors at the Student Health
Center of Colorado State University (Fort Collins, CO). PBMC were isolated from heparinized venous blood by separation on a Ficoll-Hypaque
gradient (32). The cells were washed and resuspended at 1 ⫻ 106/ml in
RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 10 ␮g/ml gentamicin, and 10% heat-inactivated AB human serum (Pel-Freez Clinical Systems, Brown Deer, WI).
cultures were maintained at 37°C, 5% CO2 during the assay. At 18 h, the
wells were pulsed with 1 ␮Ci/well [3H]TdR for 8 h before harvesting and
scintillation counting. Preincubations of IL-2 with the T cell inhibitory
protein (TIP)-containing fraction were done in polypropylene tubes for 90
min in 10 mM HEPES (pH 7.3), 150 mM NaCl. The TIP fraction of tick
saliva was obtained by gel filtration HPLC of 400 ␮l of saliva (360 ␮g
protein) on a Bio-Rad Bio-Sil size exclusion 125-5 column (125-0475;
Bio-Rad, Richmond, CA) that was perfused in 10 mM HEPES (pH 7.3) and
150 mM NaCl. Parameters of the HPLC were essentially as described
previously (37).
The Journal of Immunology
4321
biotinylated cytokine was determined by graphical analysis of the slope of
the linear data range in the quantitative competitive ELISAs, described
above. Ratios of the slopes were used to derive normalization factors for
the various cytokines. The normalization factors relative to the signal generated by bio-rmIL-2, which was set at a value of 1, were determined to be
1.81⫻ for bio-rhIL-2 and 1.65⫻ for bio-rmIFN-␥. Thus, the specific activity of the bio-IFN-␥ and bio-rhIL-2 are nearly equivalent, whereas the
bio-rmIL-2 specific activity is greater than either of the other reagents.
In the competitive binding assay, 2 ␮l of tick saliva was immobilized in
each well of the assay plate as above. Bio-rmIL-2 was premixed with
unlabeled rmIL-2 at the concentrations indicated in the Results and Fig. 4
in 5% FCS supplemented with 1⫻ PBS (pH 7.4) in polypropylene wells.
These samples were transferred to the plates with immobilized saliva and
incubated for 3 h at room temperature. The assay was completed as were
the noncompetitive assays. The positive control for bio-IFN-␥ was done
using the plate-bound capture Ab for the IFN-␥ ELISA (described above).
Trypsin digestion of saliva
FIGURE 1. The ability of I. scapularis saliva to inhibit T cell proliferation can be washed away after several hours of preincubation with splenocytes. C57BL/6 SC were preincubated for the indicated times with saliva;
controls received no saliva. After being preincubated, the samples were
either washed with culture medium or were left unwashed, plated in triplicates (5 ⫻ 105 SC/well) in microtiter wells, and then stimulated with
soluble anti-CD3 mAb (control wells received no mitogenic stimulation).
After 24 h, the wells were pulsed with [3H]TdR for 18 h and then harvested. T cell proliferation was assessed by scintillation counting. Presented results are the mean ⫾ SD of triplicate cultures.
Statistics
The human proliferation assays were analyzed by ANOVA and the Student-Newman-Keuls method for pairwise multiple comparisons. Values of
p ⬍ 0.05 were accepted as indicating a significant difference between treatments. Other experiments were run a minimum of three times, and the
results of representative experiments are graphed as the mean of triplicate
samples ⫾ SD.
Results
The T cell proliferation inhibitory activity of I. scapularis saliva
can be washed away after several hours of preincubation with
splenocytes
The simplest apparent model for the T cell inhibitory capacity of
tick saliva would be a mechanism dependent upon a direct interaction of a salivary factor(s) with T cells to mediate suppressive
effects on T cell proliferation. Previous work established that preincubation of saliva with SC was not necessary to see the inhibitory effect of saliva (24). This observation was extended by testing
the ability of tick saliva to mediate its effect on SC if the cells were
preincubated with saliva for several hours and then washed extensively to remove saliva proteins that were not tightly interacting
with cells before mitogenic stimulation. The data depicted in Fig.
1 show that the ability to inhibit T cell activity can be washed away
from SC even after 2– 4 h of preincubation before stimulation.
From looking at the results with the unwashed saliva-treated samples, it is clear that the ability to inhibit is still present even after
4 h of preincubation. These results suggest that, although saliva
must be present to mediate a suppressive effect on proliferation, a
direct and avid interaction with T cells does not occur.
I. scapularis saliva inhibits detection of mouse IL-2 in an ELISA
The previous experiment presented the possibility that the salivary
T cell inhibitor does not interact directly with T cells. Working
with this information and the results of previous studies showing
decreases in the amount of IL-2 present in culture supernatants of
Con A-stimulated SC that were treated with saliva (24), we hypothesized that saliva was binding IL-2. We tested this using a
mouse IL-2-specific ELISA, as shown in Fig. 2A. For this exper-
iment, tick saliva was preincubated with mouse IL-2 before being
transferred to microtiter wells coated with the IL-2 capture Ab. As
depicted in Fig. 2A, the detection of IL-2 in this ELISA is diminished in a dose-dependent fashion both in terms of the amount of
saliva and the amount of IL-2 present. This result supports the
presence of an IL-2 binding factor in tick saliva that blocks one or
both of the epitopes recognized by the matched anti-IL-2 mAbs
resulting in decreased IL-2 detection in the ELISA. This activity
was not influenced by the glycosylation state of the mouse IL-2,
because detection of both rIL-2 produced in Escherichia coli and
native IL-2 from SC culture supernatants were inhibited (data not
shown).
A control experiment consisting of an ELISA specific for mouse
IFN-␥ was run using the same batch of saliva to test the specificity
of the IL-2 binding factor. This T cell cytokine was chosen as a
control because its production has also been shown to be markedly
decreased by in vitro-stimulated lymphocytes in the presence of
SGE (19, 22, 23, 30, 40). As shown in Fig. 2B, tick saliva does not
affect the detection of rmIFN-␥ in a dose-dependent manner, illustrating the specificity of the IL-2 binding activity defined in Fig.
2A and also that I. scapularis saliva does not appear to have IFN-␥
binding capacity under the conditions tested.
I. scapularis saliva binds both mouse and human IL-2
To directly verify the IL-2 binding capacity and specificity of the
I. scapularis IL-2 binding activity, tick saliva was plated on microtiter wells, and then its ability to bind bio-IL-2 was determined.
Although mouse and human IL-2 sequences are 63% conserved at
the amino acid level (41), they are not fully biologically interchangeable (42). Thus, the ability of saliva to bind both mouse and
human IL-2 was examined. The specificity for IL-2 was again
tested by the inclusion of an IFN-␥ control. The results of these
experiments are shown in Fig. 3. The graphed data show that plateimmobilized saliva binds bio-rmIL2 and bio-rhIL-2 in a dose-dependent manner and at essentially equivalent levels. This result
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An aliquot of I. scapularis saliva was mixed with an equal volume of 1
mg/ml trypsin prepared in 10 mM Tris-HCl and 5 mM CaCl2 (pH 8). The
negative control was prepared with water in place of saliva, and the positive control was prepared with 10 mM Tris-HCl (pH 8) and 5 mM CaCl2
without trypsin. Trypsin-only and saliva-only controls were also prepared.
All samples were incubated for 24 h at 37°C and then stopped with a 2⫻
mass excess (relative to trypsin) of 10 mg/ml SBTI prepared in dH2O. An
equivalent volume of dH2O was added to samples that were not treated
with SBTI. One trypsin-only sample was combined with the saliva-only
sample to generate the “saliva with trypsin plus SBTI” control. The variously treatment samples were then divided into two portions that were
tested, respectively, in the IL-2 ELISA interference assay and the platebound saliva bio-rmIL-2 binding assays described above and in Results.
4322
IDENTIFICATION OF AN IL-2 BINDING PROTEIN IN TICK SALIVA
implies a similar binding affinity for both species of IL-2. However, the saliva does not show evidence of bio-rmIFN-␥ binding,
even when up to 8 ␮l of saliva is plated.
The signal from bound bio-IL-2 titrates with the decrease in
saliva plated; thus, plate-immobilized saliva retains its ability to
bind the IL-2 ligand. The absence of significant binding to biormIFN-␥ also shows that the binding of the bio-IL-2 is not due to
biotin binding activity in tick saliva. Repeating this analysis with
different batches of saliva showed that the amount of IL-2 binding
activity varies, but is always present. This batch-to-batch variance
was also evident when the IL-2 ELISA interference assay was used
(data not shown).
The binding of bio-rmIL-2 by plated saliva can be specifically
competed away with rmIL-2
As a more direct means of assessing the specificity of the IL-2
binding capacity of the saliva, the direct plate-binding assay was
adapted to a competitive binding format. This assay was also important as a control for nonspecific signals generated by potential
protein contaminants present in the commercially obtained preparations of rmIL-2 that were biotinylated during the generation of
bio-rmIL-2. The IL-2 supplier was chosen because they do not add
any carrier protein to their recombinant cytokines. The rmIL-2 and
rhIL-2 were HPLC-purified to ⬎97%, and the rmIFN-␥ was
⬎95% pure. A constant amount of saliva was bound to each well
and then incubated with a mixture that contained a constant
amount of bio-rmIL-2 (⬃13 pg) and varying amounts of nonbiotinylated competitor rmIL-2. As illustrated in Fig. 4, the signal
from binding of bio-rmIL-2 was competed away by addition of
between 8- and 16-fold of unlabeled competitor and is essentially
the same as theoretically predicted for a competitive inhibitor with
equal affinity for the binding site. As a negative control, the assay
was also run in parallel in wells that were not plated with saliva.
The IL-2 binding factor is a protein
Because the majority of the inhibitory effect of tick saliva on T cell
proliferation has previously been shown to be due to a protein (24),
we used the same approach to test whether the IL-2 binding activity was due to a protein in the saliva. Saliva was digested with
trypsin for 24 h, the reaction was stopped with excess SBTI, and
then tested for its ability to inhibit the IL-2 ELISA and bind biormIL-2 using the assay systems described above. As shown in Fig.
5 (groups C), trypsin treatment eliminates the IL-2 binding capacity of the saliva; therefore, the IL-2 binding factor is a protein.
Control reactions showed that trypsin treated with SBTI did not
affect either in vitro assay when added alone (Fig. 5, groups D) and
that saliva was still capable of binding IL-2 when trypsin treated
with SBTI was added to each assay system (Fig. 5, groups E).
I. scapularis saliva inhibits proliferation of human T cells
Previous experimentation has established that tick saliva contains
a potent T cell proliferation inhibitor by using lymphocytes from a
variety of animal models including mice and cows. Due to the
identification of a novel cytokine binding activity in tick saliva
(described above), and because of the importance of I. scapularis
as a vector of human pathogens (11, 12, 15), we tested the ability
of tick saliva to inhibit human T cell proliferation. For these experiments, PBMCs were isolated from healthy human donors and
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FIGURE 2. I. scapularis saliva inhibits detection of mouse IL-2 in an
ELISA. A, Various concentrations of saliva were preincubated for 3 h with
various amounts of rmIL-2, as indicated, before addition of the mixtures to
capture-Ab-coated wells. The assay was completed as described in Materials and Methods. B, Same as A, except preincubation of saliva was with
rmIFN-␥ and the ELISA was IFN-␥-specific. The same batch of saliva was
used for both depicted experiments.
FIGURE 3. I. scapularis saliva binds both mouse and human IL-2. Various amounts of saliva, as indicated, were immobilized in wells and then
incubated with an excess of either bio-rmIL-2, bio-rhIL-2, or bio-rmIFN-␥
for 4 h. A blank control of saliva-coated wells processed without a biotinylated cytokine incubation step was also run. Binding of the cytokines
was then detected with avidin-HRP. The values plotted for bio-rhIL-2 and
bio-rmIFN-␥ absorbance are normalized relative to bio-rmIL-2 to account
for differences in the degree of biotinylation of the cytokines (see Materials
and Methods). Results are presented as the mean ⫾ SD of triplicate cultures with blank absorbance values subtracted. The positive control for
bio-rmIFN-␥ was done with a plate-bound IFN-␥ capture Ab.
The Journal of Immunology
4323
stimulated with the T cell mitogenic lectin, PHA. As is evident in
Fig. 6, proliferation of T cells from all six donors was diminished
from 52– 88% when treated with a 1:100 dilution of saliva ( p ⬍
0.05), and the effect of the saliva could be diluted away, as the
difference between each of the three treatment groups is significant
( p ⬍ 0.05).
Saliva inhibits growth of an IL-2-dependent cell line
To assess the suggested mechanism of TIP as an IL-2 binding
protein (IL-2BP), the effects of the saliva on the growth of the
IL-2-dependent cell line CTLL-2 were tested. As CTLL-2 cells can
be stimulated with both human and mouse IL-2 (36), this approach
allows the hypothesis to be addressed with a bioassay system. It
was found in preliminary experiments that CTLL-2 cells are very
sensitive to even low concentrations of PGE2, which is present in
tick saliva at relatively high levels (24, 43). Thus, to obtain TIP
without contaminating PGs, the TIP-containing fraction of I.
scapularis saliva was purified using gel filtration HPLC. All three
assays for TIP (descibed above) showed that the TIP activity cofractionated with an apparent mass of ⬃60 kDa (data not shown).
As shown in Fig. 7A, proliferation of CTLL-2 cells grown in the
presence of 10 pg of human or mouse IL-2 are inhibited in a
dose-dependent manner by TIP.
If the inhibition of CTLL-2 proliferation is indeed the result of
IL-2BP neutralization of growth factor activity of IL-2, it should
be possible to negate this effect by adding extra IL-2 to the assay.
This experiment was done by preincubating IL-2 and TIP before
addition to the CTLL-2 cells. Results for the mouse and human
IL-2 experiments are shown in Fig. 7B, where this prediction is
fulfilled.
Discussion
The activation of T cells plays an important role in the orchestration of both humoral and cellular immune responses. Although a
large body of evidence supports the existence of nonspecific T cell
inhibitory activities in the saliva of several species of ixodid ticks
(16, 20, 23–29), with one exception (29), definitive experiments
aimed at elucidating the mechanism of T cell proliferation inhib-
FIGURE 5. The IL-2 binding factor is a protein. I. scapularis saliva was
digested with trypsin, and the reaction was stopped by addition of excess
SBTI (see Materials and Methods). To determine whether this treatment
eliminated the ability of the saliva to bind IL-2, the digests were tested in
the plate-bound saliva bio-rmIL-2 binding assay (A) and the IL-2 capture
ELISA interference assay (B). The treatment groups were as follows: group
A, positive control of undigested saliva; group B, negative control of no
saliva; group C, saliva digested with trypsin for 24 h and then stopped with
excess SBTI; group D, trypsin treated with excess SBTI; and group E,
trypsin treated with SBTI then added to saliva.
itory factors have been lacking. Toward this end, Urioste et al. (24)
demonstrated that the major T cell inhibitory activity in I. scapularis saliva is proteinaceous due to its inactivation by trypsin treatment. Bergman et al. have also recently identified a 36-kDa protein
in tick SGE of Dermacentor andersoni that inhibits T cell proliferation (26, 28).
One of the hallmarks of T cell activation is the production of
IL-2, which serves as an autocrine growth factor (44). Thus, it is
not surprising that many studies have also identified a decrease in
IL-2 production by lymphocytes exposed to tick SGE or saliva in
vitro (16, 23, 24, 27) or in ex vivo-restimulated lymphocytes from
tick-infested animals (20, 22). The full importance of this observation is brought into perspective when it is considered that IL-2
also acts as a paracrine growth factor and signaling molecule to
activated T cells and other immune effector cells that express IL-2
receptors on their surface, including B cells, NK cells, CTLs, lymphokine-activated killers, monocytes, and macrophages (44 – 46).
Thus, the nonspecific effect of tick saliva on T cell activation has
potentially far-reaching consequences in the generation of anti-tick
immunity and pathogen transmission. Of interest in this regard, it
has been observed that the functions of NK cells (47, 48) and
macrophages (24, 27) are also inhibited by tick saliva.
In the search for a mechanism of the T cell inhibitory action of
tick saliva, we hypothesized that the salivary factor(s) responsible
for inhibiting T cell proliferation interacted directly with T cells.
The ability to wash away saliva without residual inhibition of proliferation demonstrated that this hypothesis was probably incorrect. Although the results do not eliminate the possibility that T
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FIGURE 4. The binding of bio-rmIL-2 by plated saliva can be specifically competed away with rmIL-2. A constant amount of saliva (2 ␮l) was
immobilized in wells and then incubated for 2 h with mixtures containing
a constant concentration of bio-rmIL-2 and various amounts of unlabeled
competitor rmIL-2 (1⫻ competitor, ⬃13 pg). Control wells were prepared
without immobilized saliva. The assay was completed as in Fig. 3. Results
are presented as the mean of triplicate samples ⫾ SD. As a reference, the
expected inhibition of a theoretical competitive inhibitor with equal affinity
for the binding site is also graphed using the equation: OD ⫽ y (1/(x ⫹ 1)),
where y ⫽ OD without competitor and x ⫽ fold of competitor.
4324
IDENTIFICATION OF AN IL-2 BINDING PROTEIN IN TICK SALIVA
cell inhibitors interact directly with T cells, they indicate that any
interactions are likely to be weak or to occur after T cell activation.
Furthermore, this experiment also illustrates the temporal requirement for the presence of saliva during and after mitogenic stimulation of SC to mediate its effect on the in vitro proliferation of T
cells. This result led us to test whether tick saliva was directly
interfering with IL-2 activity using an IL-2-specific ELISA. The
results of this analysis were in keeping with the observation that
the presence of saliva was necessary for the inhibited proliferation
to be evident. T cells produce IL-2 when they are stimulated, so the
IL-2BP cannot function until after T cell activation. Previous studies have also shown that IFN-␥ production is decreased by tick
SGE exposure (23, 30, 40) or tick infestations (19, 22). Although
our analysis shows that I. scapularis saliva does not appear to bind
IFN-␥ under the tested conditions, it is known that IL-2 induces T
cells and NK cells to produce IFN-␥ (49). Thus, it is possible that
the IL-2BP is responsible for several identified effects of tick saliva
on host immune effector cells; however, clarifying this will require
further studies that address causal linkages between the observed
phenomena.
The identification of Ig binding proteins (IGBPs) in the saliva of
several species of ixodid ticks (50, 51) raises the caveat that the
inhibition of the IL-2 ELISA is due to IGBP (though this activity
has not been reported in I. scapularis). The lack of IFN-␥ ELISA
inhibition by the saliva dispels this concern. However, because the
isotypes of the matched pairs of rat IgG anti-cytokine mAbs used
in the ELISAs were not all the same, the ability of plate-bound I.
scapularis saliva to bind each of the mAbs used was tested (see
Materials and Methods). This experiment showed that there was
not significant binding of any of the rat mAbs by plate-bound
saliva (data not shown). Thus, IGBP activity is not a concern in the
interpretation of the results of the experiments depicted in Fig. 2.
Most previous analyses of the immunomodulatory effects of tick
saliva have focused on animal models, leaving the relevance to
humans unaddressed. It has been suggested that ectoparasites are
closely adapted to their hosts (5) so that they can counteract their
hosts’ defenses. This idea has also been used to support observations that more robust resistance to tick feeding has typically been
FIGURE 7. Saliva inhibits growth of an IL-2-dependent cell line.
CTLL-2 cells were washed and plated at 5000 cells/well in the presence of
human or mouse IL-2 at 10 pg/well. A, Various amounts of the TIP fraction
of saliva were added to the wells, which were incubated for 20 h and then
pulsed for 8 h with [3H]TdR before the proliferative response was measured by harvesting and scintillation counting. Results are presented as the
mean of triplicate samples ⫾ SD. B, Saliva was preincubated with various
amounts of mouse or human IL-2, as indicated, before addition to CTLL-2
cells and completion of the assay as described above.
associated with unnatural tick-host associations (5, 21). In one report, Fuchsberger et al. demonstrated that Rhipicephalus appendiculatus SGE inhibits expression of several cytokine mRNAs in
LPS-stimulated human PBMCs (40). Although they were not able
to detect IL-2 mRNA, they did recognize that SGE effects on
cytokine expression levels were dependent on stimulation of the
PBMCs. Due to the significance of I. scapularis as a vector for
several emerging human diseases, we included experiments that
showed that tick saliva binds not only to mouse IL-2, but also to
human IL-2 with similar affinities. Interspecies cross-reactivity of
the IL-2BP points to the likelihood that the binding site on IL-2 is
a conserved region or feature of the molecule. Furthermore, we
used PBMCs from human donors to show that I. scapularis saliva
acts as an inhibitor of human T cell proliferation. This finding was
further supported by the use of a CTLL-2 cell bioassay to show
that binding of IL-2 and neutralization of its growth factor activity
appears to be the mechanism responsible for inhibition of both
human and mouse T cell proliferation. These experiments support
the idea that tick saliva may play a role in the transmission of
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FIGURE 6. I. scapularis saliva inhibits proliferation of human PBMCs.
Saliva at 1:100 and 1:200 final dilution was added to 2 ⫻ 105 PBMC/well
from six different human donors and stimulated with 2 ␮g/ml PHA. At 24 h
after stimulation, the cells were pulsed for 18 h with [3H]TdR. Cultures
were interrupted by harvesting, and the proliferative response was measured by scintillation counting. Results are presented as the mean of triplicate samples; error bars were left off of data points for clarity. Proliferation after treatment with both saliva dilutions is significantly different than
the untreated (no saliva, “none”) control samples and than each other (p ⬍
0.05). The mean value for each treatment group is indicated by a bar.
The Journal of Immunology
festations of the host with uninfected nymphal ticks before challenge with infected nymphs. However, this approach did not result
in protection when using a mouse model for I. ricinus (63). Despite
variable success, these studies provide the impetus for the pursuit
of tick Ags that can protect the host from ticks and tick-borne
pathogens.
The identification of a secreted IL-2 binding factor is quite novel
for an ectoparasite system, and, to the best of our knowledge, the
IL-2BP in I. scapularis saliva represents the first evidence for direct IL-2 targeting by any parasite or pathogen of mammals. Various means of subverting the immune system of hosts through their
cytokines and chemokines have evolved in several classes of
pathogens, although none of these directly target IL-2 (64). It has
been suggested that no viral or bacterial pathogens have directly
targeted IL-2 because the general immunosuppression that would
ensue might give other pathogens a chance to successfully compete against them (64). Perhaps ectoparasites such as I. scapularis
can succeed with the strategy of direct subversion of a central
signaling cytokine because they do not multiply within the host.
Alternatively, if the effect of tick saliva on host immune function
is predominantly local rather than systemic, direct IL-2 targeting
may not adversely effect the entire immune system of the host. In
either case, this strategy may be important to the effectiveness of
ticks as disease vectors.
The IL-2BP could be important to the potentiation of the feeding
site for transmission of pathogens by aiding establishment of the
infection. Indeed, the saliva of the vector should not only be considered a pharmacological milieu that allows for blood to be drawn
over a protracted period and a vehicle for the transmission of
pathogens, but also as an important vector competence factor (9).
Although we have functionally identified an IL-2BP in I. scapularis that may provide the mechanism for the inhibition of T cell
proliferation by tick saliva, it will now be useful to know whether
this IL-2 binding capacity is found in other tick species or other
blood-feeding ectoparasites. Furthermore, is this the only example
of cytokine-targeted host immunomodulation present in arthropods
or other ectoparasites? This discovery can be seen as another example of how well evolved ectoparasite-host interactions appear to
be. Thus, the simple and elegant mechanism of tick salivary IL2BP, the sequestration of IL-2, has multiple potential effects on the
quality and specificity of the immune response generated to any
foreign Ags present during the feeding process, including those of
pathogens introduced at the feeding site. Given the nature of this
activity’s assault on the immune system of the host, the discovery
of this mechanism also raises important issues regarding the development of vaccines against ticks, an idea that has gained much
support because it holds the potential of blocking transmission of
several pathogens with a single vaccination (7, 8). The IL-2BP
may inhibit the effectiveness of this approach and, thus, may turn
out to be an important target that must be overcome for this strategy to be successful.
Acknowledgments
We are grateful to Dr. C. Brodskyn for assistance with the human cell
work, as well as to Marc Keen and Dr. John Belisle for expert help with
HPLC. We thank Drs. M. L. Mbow, G. K. DeKrey, C. Brodskyn, and N.
Zeidner for helpful discussions of this research and critical review of the
manuscript.
References
1. Titus, R. G., and J. M. C. Ribeiro. 1988. Salivary gland lysates from the sand fly
Lutzomyia longipalpis enhance Leishmania infectivity. Science 239:1306.
2. Jones, L. D., W. R. Kaufman, and P. A. Nuttall. 1992. Modification of the skin
feeding site by tick saliva mediates virus transmission. Experientia 48:779.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
diseases to humans and that the same phenomena identified in
animal models are likely to occur in humans exposed to tick bites.
The studies reported here were done with saliva from adult ticks,
whereas, in the field, nymphal ticks are the primary threat for disease transmission to humans. Studies of saliva and SGE of nymphs
are exceedingly challenging due to the difficulty of obtaining sufficient amounts of material for detailed analysis. However, we
have done preliminary experiments that verified that T cell proliferation is inhibited by nymph SGE (R. D. Gillespie, unpublished
data).
Support for the proposed IL-2 binding mechanism of inhibited T
cell proliferation is provided indirectly by other studies. Zeidner et
al. demonstrated that the passive administration of IL-2 to mice
during challenge feeding by I. scapularis nymphs infected with the
Lyme disease spirochete, Borrelia burgdorferi, resulted in 50%
protection from infection (52). In studies of the effects of passive
rhIL-2 administration to rabbits during infestation with Ixodes ricinus adults, the European vector of Lyme disease spirochetes, it
was found that the rabbits were more resistant to subsequent tick
infestations and developed dramatically stronger delayed-type hypersensitivity (DTH) reactions against SGE (53). Furthermore, the
development of DTH to SGE occurred in the IL-2-treated animals
after only one infestation, compared with the multiple infestations
usually necessary for DTH development to SGE of I. ricinus (54).
The importance of IL-2 to this phenomenon was established by
showing that cyclosporin A treatment resulted in attenuation of the
DTH response to SGE in rabbits (54). In a similar study, it was
shown that acquired resistance to I. ricinus by the bank vole (a
natural host for the ectoparasite) could be partially disrupted by
cyclosporin A treatment (55).
Experiments using I. ricinus infestations of mice have shown
that the effects of tick saliva are mediated at the attachment site and
the lymph nodes draining the area (17–21), although systemic effects have also been documented with I. scapularis (22, 56). The
IL-2 ELISA inhibition data show that 1 ␮l of saliva is capable of
binding ⬃4 pg of IL-2, although we routinely measure binding
capacities ranging between ⬃1 and 6 pg/␮l in different batches of
saliva using this assay. This variability is not surprising, because
the ticks are not truly synchronized in their feeding/salivation state
when they are removed from the host for saliva collection. When
it is considered that, on average, a nearly replete adult I. scapularis
female can be induced to secrete 10 ␮l of saliva during a 1- to 2-h
period after being removed from the host (R. D. Gillespie, unpublished data), and the tick feeds for 5– 8 days salivating intermittently, it is quite possible for IL-2BP to be present at levels likely
to have marked effects on the immune system of the host. At the
local level of the draining lymph node, IL-2 sequestration by IL2BP would effect the proliferation of naive T cells and Th1 cells,
as Th2 cells are not dependent on IL-2 for expansion after Ag
stimulation (57, 58). This scenario fits nicely with the generalized
model of Th1 cell and inflammation suppression and Th2 cell enhancement caused by tick infestations (19 –23). It is known that
starvation for IL-2 in activated T cells can lead to anergy of the
activated cells (59, 60); thus, IL-2BP may adversely effect the
ability of a host to develop adaptive immunity to ticks and tickborne pathogens. The full biological consequences of this activity
are likely to depend on the issue of scale, both with respect to the
developmental stage of the tick, i.e., larvae, nymph, or adult, and
degree of infestation, e.g., a heavy infestion on a small animal vs
one or a few ticks on a large animal.
It is important to note that recent studies using mice (61) and
guinea pigs (62) have indicated that it is possible to generate adaptive immunity to I. scapularis that also results in the blocking of
transmission of B. burgdorferi. This strategy required multiple in-
4325
4326
IDENTIFICATION OF AN IL-2 BINDING PROTEIN IN TICK SALIVA
34. Stafford, K. C. I., A. J. Denicola, and L. A. Magnarelli. 1996. Presence of Ixodiphagus hookeri (Hymenotera: Encyrtidae) in two Connecticut populations of
Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 33:183.
35. Ribeiro, J. M. C., and A. Spielman. 1986. Ixodes dammini: salivary anaphylatoxin
inactivating activity. Exp. Parasitol. 62:292.
36. Davis, L. S., P. E. Lipsky, and K. Bottomly. 1995. Measurement of human and
murine interleukin 2 and interleukin 4. In Current Protocols in Immunology.
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober,
eds. John Wiley & Sons, Inc., New York, p. 6.3.1.
37. Ribeiro, J. M. C., and T. N. Mather. 1998. Ixodes scapularis: salivary kininase
activity is a metallo dipeptidyl carboxypeptidase. Exp. Parasitol. 89:213.
38. Lisanti, M. P., and M. Sargiacomo. 1995. Biotinylation and analysis of membrane-bound and soluble proteins. In Current Protocols in Immunology.
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober,
eds. John Wiley & Sons, Inc., New York, p. 8.16.1.
39. Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring
Harbor Lab. Press, New York, p. 586.
40. Fuchsberger, N., M. Kita, V. Hajnicka, J. Imanishi, M. Labuda, and P. A. Nuttall.
1995. Ixodid tick salivary gland extracts inhibit production of lipopolysaccharideinduced mRNA of several different human cytokines. Exp. Appl. Acarol. 19:671.
41. Kashima, N., C. Nishi-Takaoka, T. Fujita, S. Taki, G. Yamada, J. Hanuro, and
T. Tanaguchi. 1985. Unique structure of murine interleukin-2 as deduced from
cloned cDNAs. Nature 313:402.
42. Mosmann, T. R., T. Yokota, R. Kastelein, S. M. Zurawski, N. Arai, and
Y. Takebe. 1987. Species-specificity of T cell stimulating activities of IL-2 and
BSF-1 (IL-4): comparison of normal and recombinant, mouse and human IL-2
and BSF-1 (IL-4). J. Immunol. 138:1813.
43. Bowman, A. S., J. W. Dillwith, and J. R. Sauer. 1996. Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12:388.
44. Smith, K. A. 1992. Interleukin-2. Curr. Opin. Immunol. 4:271.
45. Theze, J., P. M. Alzari, and J. Bertoglio. 1996. Interleukin 2 and its receptors:
recent advances and new immunological functions. Immunol. Today 17:481.
46. Siegel, J. P., M. Sharon, P. L. Smith, and W. J. Leonard. 1987. The IL-2 receptor
␤ chain (p70): role in mediating signals for LAK, NK, and proliferative activities.
Science 238:75.
47. Kopecky, J., and M. Kuthejlova. 1998. Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro. Parasite Immunol. 20:169.
48. Kubes, M., N. Fuchsberger, M. Labuda, E. Zuffova, and P. A. Nuttall. 1994.
Salivary gland extracts of partially fed Dermacentor reticulatus ticks decrease
natural killer cell activity in vitro. Immunology 82:113.
49. Trinchieri, G., and B. Perussia. 1985. Immune interferon: a plieotropic lymphokine with multiple effects. Immunol. Today 6:131.
50. Wang, H., and P. A. Nuttall. 1994. Excretion of host immunoglobulin in tick
saliva and detection of IgG-binding proteins in tick haemolymph and salivary
glands. Parasitology 109:525.
51. Wang, H., and P. A. Nuttall. 1995. Immunoglubulin-G binding proteins in the
ixodid ticks, Rhipicephalus appendiculatus, Amblyomma variegatum and Ixodes
hexagonus. Parasitology 111:161.
52. Zeidner, N., M. Dreitz, D. Belasco, and D. Fish. 1996. Suppression of acute
Ixodes scapularis-induced Borrelia burgdorferi infection using tumor necrosis
factor-␣, interleukin-2, and interferon-␥. J. Infect. Dis. 173:187.
53. Schorderet, S., and M. Brossard. 1994. Effects of human recombinant interleukin-2 on resistance, and on the humoral and cellular response of rabbits infested
with adult Ixodes ricinus ticks. Vet. Parasitol. 54:375.
54. Girardin, P., and M. Brossard. 1989. Effects of cyclosporin-A on the humoral
immunity to ticks and on cutaneous immediate and delayed hypersensitivity reactions to Ixodes ricinus L. salivary-gland antigens in re-infested rabbits. Parsitol. Res. 75:657.
55. Dizij, A., and K. Kurtenbach. 1995. Clethrionomys glareolus, but not Apodemus
flavicollis, acquires resistance to Ixodes ricinus L., the main European vector of
Borrelia burgdorferi. Parasite Immunol. 17:177.
56. Schoeler, G. B., S. A. Manweiler, and S. K. Wikel. 1999. Ixodes scapularis:
effects of repeated infestations with pathogen-free nymphs on macrophage and T
lymphocyte cytokine responses of BALB/c and C3H/HeN mice. Exp. Parasitol.
92:239.
57. Romagnani, S. 1997. The Th1/Th2 paradigm. Immunol. Today 18:263.
58. Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell
76:241.
59. DeSilva, D. R., K. B. Urdahl, and M. K. Jenkins. 1991. Clonal anergy in induced
in vitro by T cell receptor occupancy in the absence of proliferation. J. Immunol.
147:3261.
60. Schwartz, R. H. 1996. Models of T cell anergy: is there a common molecular
mechanism? J. Exp. Med. 184:1.
61. Wikel, S. K., R. N. Ramachandra, D. K. Bergman, T. R. Burkot, and J. Piesman.
1997. Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infect.
Immun. 65:335.
62. Nazario, S., S. Das, A. M. deSilva, K. Deponte, N. Marcantonio, J. F. Anderson,
D. Fish, E. Fikrig, and F. S. Kantor. 1998. Prevention of Borrelia burgdorferi
transmission in guinea pigs by tick immunity. Am. J. Trop. Med. Hyg. 58:780.
63. Mbow, M. L., M. Christe, B. Rutti, and M. Brossard. 1994. Absence of acquired
resistance to nymphal Ixodes ricinus ticks in BALB/c mice developing cutaneous
reactions. J. Parsitol. 80:81.
64. Marrack, P., and J. Kappler. 1994. Subversion of the immune system by pathogens. Cell 76:323.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
3. Cupp, E. W., and M. S. Cupp. 1997. Black fly (Diptera: Simuliidae) salivary
secretions: importance in vector competence and disease. J. Med. Entomol. 34:
87.
4. Zeidner, N. S., S. Higgs, C. M. Happ, B. J. Beaty, and B. R. Miller. 1999.
Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible
mice: an effect mimicked by injection of sialokinins, but not demonstrated in
flavivirus resistant mice. Parasite Immunol. 21:35.
5. Ribeiro, J. M. C. 1989. Role of saliva in tick/host interactions. Exp. Appl. Acarol.
7:15.
6. Ribeiro, J. M. C. 1995. How ticks make a living. Parasitol. Today 11:91.
7. Wikel, S. K., and D. Bergman. 1997. Tick-host immunology: significant advances and challenging opportunities. Parasitol. Today 13:383.
8. Gillespie, R. D., M. L. Mbow, and R. G. Titus. 2000. The immunomodulatory
factors of bloodfeeding arthropod saliva. Parasite Immunol. 22:319.
9. Bowman, A. S., L. B. Coons, G. R. Needham, and J. R. Sauer. 1997. Tick saliva:
recent advances and implications for vector competence. Med. Vet. Entomol.
11:277.
10. Balashov, Y. S. 1972. Bloodsucking ticks (Ixodoidea): vectors of diseases of man
and animals. Misc. Publ. Entomol. Soc. Am. 8:161.
11. Centers for Disease Control and Prevention. 1997. Lyme disease—United States,
1996. Morbid. Mortal. Wkly. Rep. 46:531.
12. Magnarelli, L. A., J. S. Dumler, J. F. Anderson, R. C. Johnson, and E. Fikrig.
1995. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J. Clin. Microbiol. 33:3054.
13. Piesman, J., T. N. Mather, S. R. I. Telford, and A. Spielman. 1986. Concurrent
Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini.
J. Clin. Microbiol. 24:446.
14. Telford, S. R., III, J. E. Dawson, P. Katavolos, C. K. Warner, C. P. Kolbert, and
D. H. Persing. 1996. Perpetuation of the agent of human granulocytic ehrlichiosis
in a deer tick-rodent cycle. Proc. Natl. Acad. Sci. USA 93:6209.
15. Walker, D. H. 1998. Tick-transmitted infectious diseases in the United States.
Annu. Rev. Public Health 19:237.
16. Ribeiro, J. M. C., G. T. Makoul, J. Levine, D. R. Robinson, and A. Spielman.
1985. Antihemostatic, antiinflammatory, and immunosuppressive properties of
the saliva of a tick, Ixodes dammini. J. Exp. Med. 161:332.
17. Mbow, M. L., B. Rutti, and M. Brossard. 1994. Infiltration of CD4⫹ CD8⫹ T
cells, and expression of ICAM-1, Ia antigens, IL-1␣ and TNF-␣ in the skin lesion
of BALB/c mice undergoing repeated infestations with nymphal Ixodes ricinus
ticks. Immunology 82:596.
18. Mbow, M. L., B. Rutti, and M. Brossard. 1994. IFN-␥, IL-2, and IL-4 mRNA
expression in the skin and draining lymph nodes of BALB/c mice repeatedly
infested with nymphal Ixodes ricinus ticks. Cell. Immunol. 156:254.
19. Ganapamo, F., B. Rutti, and M. Brossard. 1995. In vitro production of interleukin-4 and interferon-␥ by nymph node cells from BALB/c mice infested with
nymphal Ixodes ricinus ticks. Immunology 85:120.
20. Ganapamo, F., B. Rutti, and M. Brossard. 1996. Cytokine production by lymph
node cells from mice infested with Ixodes ricinus ticks and the effect of tick
salivary gland extracts on IL-2 production. Scand. J. Immunol. 44:388.
21. Brossard, M., and S. K. Wikel. 1997. Immunology of interactions between ticks
and hosts. Med. Vet. Entomol. 11:270.
22. Zeidner, N., M. L. Mbow, M. Dolan, R. Mussung, E. Baca, and J. Piesman. 1997.
Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host
immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice. Infect. Immun. 65:3100.
23. Ramachandra, R. N., and S. K. Wikel. 1992. Modulation of host-immune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine production. J. Med. Entomol. 29:818.
24. Urioste, S., L. R. Hall, S. R. Telford, I. I. I., and R. G. Titus. 1994. Saliva of the
Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism. J. Exp. Med. 180:1077.
25. Wikel, S. K. 1982. Influence of Dermacentor andersoni infestation on lymphocyte responsiveness to mitogens. Ann. Trop. Med. Parsitol. 76:627.
26. Bergman, D. K., R. N. Ramachandra, and S. K. Wikel. 1998. Characterization of
an immunosuppressant protein from Dermacentor andersoni (Acari: Ixodidae)
salivary glands. J. Med. Entomol. 35:505.
27. Ferreira, B. R., and J. S. Silva. 1998. Saliva of Rhipicephalus sanguineus tick
impairs T cell proliferation and IFN-␥-induced macrophage microbicidal activity.
Vet. Immunol. Immunopathol. 64:279.
28. Bergman, D. K., M. J. Palmer, M. J. Caimano, J. D. Radolf, and S. K. Wikel.
2000. Isolation and molecular cloning of a secreted immunosuppressant protein
from Dermacentor andersoni salivary gland. J. Parasitol. 86:516.
29. Inokuma, H., D. H. Kemp, and P. Willadsen. 1994. Prostaglandin E2 production
by the cattle tick (Boophilus microplus) into feeding sites and its effect on the
response of bovine mononuclear cells to mitogen. Vet. Parasitol. 53:293.
30. Ramachandra, R. N., and S. K. Wikel. 1995. Effects of Dermacentor andersoni
(Acari: Ixodidae) salivary gland extracts on Bos indicus and B. taurus lymphocytes and macrophages: in vitro cytokine elaboration and lymphocyte blastogenesis. J. Med. Entomol. 32:338.
31. Swain, S. L. 1991. Lymphokines and the immune response: the central role of
interleukin-2. Curr. Opin. Immunol. 3:304.
32. Goldroen, M. H., P. J. Gannon, M. Lutz, and E. D. Holyoke. 1977. Isolation of
human peripheral blood lymphocytes: modification of a double discontinuous
density gradient of Ficoll-Hypaque. J. Immunol. Methods 14:15.
33. Leo, O., M. Foo, D. H. Sachs, L. E. Samelson, and J. A. Bluestone. 1987. Identification of a monoclonal antibody specific for murine T3 polypeptide. Proc.
Natl. Acad. Sci. USA 84:1374.