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Division of Immunology and Transplantation Biology, Department of Pediatrics, and
Department of Pathology, Stanford University, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis. The inhibitory
effect of HKL on AHR depended on the presence of IL-12 and
CD8+ T cells and was associated with an increase of IL-18
mRNA expression. Thus, our results demonstrate that HKL as an adjuvant
for immunotherapy mediates immune deviation from a pathological
Th2-dominated response toward a protective immune response in
peripheral lymphoid tissues and in the lungs and may be clinically
effective in the treatment of patients with established asthma and
allergic disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although our understanding of the inflammatory process in asthma has improved remarkably over the past decade, the prevalence of asthma in industrialized countries has increased by almost 80% since 1980 (6). The specific causes for this increase in prevalence are not clear (7), but the rise in prevalence may be due in part to the absence of effective therapies that reverse the progression of, or cure, this disease. Currently available therapies, such as inhaled corticosteroids, anti-leukotrienes, or ß2-agonists, focus rather on symptom relief, reduction or neutralization of effector molecules, and inflammatory mediators. This approach, although effective for acute disease and for relieving symptoms, however, has limited long-term salutary effects because the environmental factors that cause and precipitate asthma are not eliminated, and patients redevelop symptoms of asthma when these medications are discontinued.
An alternative, more effective and long-lasting therapeutic approach for asthma therefore focuses on the development of vaccine strategies that alter the underlying immune response and convert or deviate detrimental allergic responses toward protective immune responses, thereby modifying the course of the disease. This immunotherapeutic strategy, however, which might cure asthma and reduce its prevalence, is feasible only if potent therapies are developed that reverse ongoing airway hyperreactivity (AHR)5 and reverse the ongoing allergic inflammatory process, which plays a critical role in the pathogenesis of asthma (5). Conventional allergen immunotherapy, while capable of reducing allergen-specific IL-4 production (8), requires multiple injections over several years time and is associated with frequent failure (9). Ag-specific methods of immunotherapy described up to now, for example using immunization with plasmids containing the cDNA for allergens (10, 11, 12), while effective in preventing the development of Th2-dominated immune responses, have not been shown to reverse ongoing AHR.
We recently demonstrated that employment of heat-killed Listeria
monocytogenes (HKL) as an immunotherapeutic adjuvant
during Ag immunotherapy successfully reversed ongoing Th2-dominated
responses and induced an Ag-specific Th1-dominated response in mice
(13). HKL, a potent stimulator of the innate immune system
and a strong inducer of IL-12 and IFN- production, greatly reduced
ongoing Ag-specific IL-4 production in lymph node (LN) cells and
reduced Ag-specific IgE production. We now show in a murine model of
asthma that HKL as an adjuvant given once with Ag prevented the
development of AHR and airway inflammation in OVA-immunized BALB/c mice
and significantly reduced airway eosinophilia and mucus production.
Moreover, when given late after allergen-sensitization, a single dose
of HKL with Ag reversed established AHR and reduced airway
inflammation. The inhibitory effect on AHR depended on the presence of
IL-12 and on CD8+ T cells, was associated with an
increase of the IL-18 mRNA expression, and required close association
between HKL and the Ag. Thus, our results demonstrate that HKL as an
adjuvant very effectively promotes protective immune responses in the
respiratory tract, and down-modulates ongoing Th2-dominated responses,
indicating that HKL as an adjuvant for allergen immunotherapy may be
clinically effective in the treatment of allergic asthma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cByJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The Stanford University Committee on Animal Welfare approved animal protocols used in this study.
Monoclonal Abs
mAbs were purified from ascites by ammonium sulfate
precipitation and ion-exchange chromatography. We used the following
hybridomas: R46A2 (anti-IFN- mAb), and 53.6.7
(anti-CD8+) obtained from the American Type
Culture Collection (Manassas, VA); XMG1.2 (anti-IFN- Ab), TRFK-4
and TRFK-5 (anti-IL-5 mAbs), generously provided by Dr. Tim Mosmann
(University of Alberta, Edmonton, Canada); BVD4-1D11 and BVD6-24G2
(anti-IL-4 mAb), generously provided by M. Howard (DNAX Research
Institute, Palo Alto, CA); and C17.8 (anti-IL12 mAb), generously
provided by G. Trinchieri (Wistar Institute, Philadelphia, PA).
Anti-38C13 Id mAb 4G10 (rat IgG2a) (14) was
obtained from S. Levy (Stanford University, Stanford, CA) and was used
as isotype control.
Immunizations
Protocol 1 (prevention of AHR).
BALB/c mice were primed in the footpads with OVA (50 µg/mouse)
adsorbed to 2 mg of alum (Al[OH]3) (Fig. 1
). Four weeks later (day 29) mice were
injected in the footpads with 200 µg OVA in IFA or with 200 µg OVA
plus 108 HKL in IFA. Mice also received 50 µg
OVA in 50 µl NaCl 0.9% intranasally on day 29. After an additional
10 days (day 39), all mice received a 100 µg booster immunization of
OVA in PBS in the footpads and were challenged with 50 µg OVA in PBS
intranasally on the same day and the 2 following days (days 40 and 41).
One day after the last intranasal challenge with OVA, AHR was measured
from conscious mice after inhalation of increasing concentrations of
methacholine in a whole body plethysmograph (day 42). After an
additional 4 days, mice were sacrificed with a lethal dose of
phenobarbital (450 mg/kg), blood was taken, bronchoalveolar lavage
(BAL) was performed, lungs were removed and fixed and lymphocytes were
isolated from the draining LN for in vitro culture (day 46).
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). AHR was measured 1 day before, and 3 days and 10
days after the injection of HKL. On day 50 mice received a final s.c.
boost with OVA (50 µg in PBS) and were sacrificed 4 days later for
BAL and lung fixation. To facilitate pulmonary aspiration during intranasal administration of Ag, mice were lightly anesthetized i.p. with 0.25 ml of ketamine (0.44 mg/ml)/xylazine (6.3 mg/ml) in normal saline. Seventy-five percent of the intranasally administered Ag can be subsequently detected in the lungs (15).
Treatment of mice with anti-cytokine Abs
BALB/c mice were injected i.p. with 1 mg of mAb C17.8 (for IL-12 depletion), mAb 53.6.7 (for CD8+ depletion), or 4G10 (rat IgG2a control) in 0.5 ml PBS 1 day before, the day of, and 3 days following immunization with OVA in IFA or OVA in IFA with HKL, according to the immunization schedule of protocol 1.
Restimulation of LN cells in vitro
Draining LN were removed and depleted of resting B cells by
adherence to goat anti-mouse Ig-coated plates. LN cells (5 x
105 cells/well) were restimulated in vitro with
OVA in DMEM (Life Technologies, Grand Island, NY), which was
supplemented as previously described (16), and contained
5 x 10-5 M 2-ME and 10% FCS (HyClone
Laboratories, Logan, UT). Cells were cultured in 96-well microtiter
plates in 150 µl medium. Supernatants were harvested after 4 days for
determination of IL-4 and IFN- levels. Cytokine content in each
sample was measured in triplicate by ELISA.
Cytokine ELISA
ELISAs were performed as previously described (17).
The Ab pairs used were as follows, listed by capture/biotinylated
detection: IL-4, BVD4-1D11/BVD6-24G2; IFN-, R4-6A2/XMG1.2.
Recombinant cytokine were used as standards, with curves generated in
1:2 dilutions from 500 to 39 pg/ml for IL-4, and from 20 to 0.156 ng/ml
for IFN-.
Measurement of anti-OVA Ab isotypes
Mice were bled at the time of sacrifice and OVA-specific Ab was measured using a modified Ag-specific ELISA. For measurement of OVA specific IgG1 and IgG2a, plates were coated overnight with 5 µg/ml OVA. After washing and blocking, serial diluted sera were added to the plates. Following overnight incubation, the plates were developed using HRP-conjugated goat anti-IgG subclass-specific Abs (Southern Biotechnology Associates, Birmingham, AL). These Abs are highly specific, are absorbed against competing subclasses, and have <1% cross-reactivity with competing subclasses (data not shown). After additional washing, OPD substrate was added, the plates developed, and the OD determined at 492 nm. Anti-OVA IgG1 mAb 6C1 and anti-OVA IgG2a mAb 3A11 (18) were used as standards for quantitation of each IgG subclass. Determination of OVA-specific IgE was performed by ELISA using rat anti-mouse IgE mAb EM95 (5.0 µg/ml) to coat plates. After the samples were applied and incubated overnight, plates were washed and biotinylated OVA (10 µg/ml) was added. Two hours later, plates were washed and HRP-conjugated streptavidin (Southern Biotechnology Associates) was added. Plates were developed with o-phenylenediamine (OPD) substrate and the OD determined at 492 nm. Sera from mice hyperimmunized with OVA in alum was used as standard for the OVA-specific IgE ELISA and was first standardized for IgE levels against an anti-OVA IgE mAb generously provided by E. Gelfand (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO) (19).
Preparation of HKL
HKL were prepared as described previously (13). A clinical isolate of Listeria monocytogenes (kindly provided by Dr. Lucy Tompkins and Barbara Allen, Stanford University) was grown in nutrient broth cultures (Difco, Detroit, MI) overnight at 37°C on a rotator. Cultures in log phase growth were harvested, centrifuged, and washed three times in PBS. The recovered bacteria were resuspended in PBS and incubated at 80°C for 1 h. After two additional washes in PBS, absence of viable colonies was confirmed by lack of growth on nutrient agar plates. Bacteria concentration was enumerated by comparing the absorbance of a serial dilution of HKL at 570 nm compared with a standard dilution of a known concentration of Listeria previously enumerated by counting the outgrowth of colonies from serial dilutions of bacteria plated on nutrient agar.
Preparation of splenic adherent cells
Spleen cells (5 x 106/ml) were cultured in cDME medium in 24-well tissue culture plates for 2–3 h at 37°C. The nonadherent cells were removed by washing with warm DMEM + 10% FCS until visual inspection revealed a lack of lymphocytes (>98% of the cell population).
RNA isolation and IL-18 RT-PCR assay
Splenic adherent cells cultured with HKL (108/ml) for 8, 16, or 24 h, and popliteal LN cells taken from mice 12 and 24 h after footpad injection of 108 HKL were analyzed for IL-18 mRNA expression. Cells were processed using Qiagen RNA isolation kits (Qiagen, Valencia, CA). Reverse transcription was performed with 200 ng of RNA, 2 µg of oligo(dT) (Life Technologies) and 1 U of Superscript II Reverse transcriptase at 60°C for 60 min. Samples were stored at -20°C until further use.
Primers specific for ß-actin and IL-18 (20) were synthesized at the Stanford PAN (Protein and Nucleic Acid) facility (Stanford, CA). cDNA (10 ng) was mixed with 10x buffer, dNTPs (0.2 mM final), MgCl2 (2.5 mM final), 5' and 3' primers, and Taq DNA polymerase (1 U/reaction; Life Technologies) in a final volume of 25 µl. PCR was performed in a DNA thermal cycler (MJ Research, Cambridge, MA) for 30 cycles, and products were visualized by electrophoresis. Data shown are representative of three experiments.
Measurement of airway responsiveness
Airway responsiveness was assessed by methacholine-induced airflow obstruction from conscious mice placed in a whole body plethysmograph (model PLY 3211, Buxco Electronics, Troy, NY). Pulmonary airflow obstruction was measured by Penh using the following formula: Penh = (Te/RT - 1) x PEF/PIF), where Penh = enhanced pause (dimensionless), Te = expiratory time, RT = relaxation time, PEF = peak expiratory flow (ml/s), and PIF = peak inspiratory flow (ml/s) (21). Enhanced pause (Penh), minute volume, tidal volume, and breathing frequency were obtained from chamber pressure, measured with a transducer (model TRD5100) connected to preamplifier modules (model MAX2270) and analyzed by system XA software (model SFT 1810, all from Buxco Electronics). Measurements of methacholine responsiveness were obtained by exposing mice for 2 min to NaCl 0.9% (Portable Ultrasonic, 5500D, DeVilbiss Health Care, Sommerset, PA), followed by incremental doses (2.5–40 mg/ml) of aerosolized methacholine and monitoring Penh. This plethysmographic method for measuring AHR has been validated after direct comparison with airway measurements in intubated mice (21).
Collection of BAL fluid and lung histology
Animals were injected i.p. with a lethal dose of phenobarbital (450 mg/kg). The trachea was cannulated, the lung was then lavaged with 0.8 ml of PBS three times, and the fluid pooled. Cells in the lavage fluid were counted using a hemocytometer, and BAL cell differentials were determined on slide preparations stained with Hansel Stain (Lide Laboratories, Florissant, MO). At least 200 cells were differentiated by light microscopy based on conventional morphologic criteria. In some animals, no BAL was performed but lungs were removed, washed with PBS, fixed in 10% formalin, and stained with hematoxylin and eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that immunization of mice with Listeria monocytogenes as adjuvant successfully biased the development of Ag-specific cytokine synthesis toward Th1 cytokine production in both primary and secondary immune responses (13). Because the pathogenesis of asthma is tightly associated with Th2 cytokines and because Th1 cytokines may protect against asthma, we investigated the ability of HKL to inhibit the development of AHR in OVA-immunized BALB/c mice.
BALB/c mice were immunized s.c. with OVA adsorbed to alum, which
provokes an OVA-specific Th2-dominated immune response. The mice were
then boosted with OVA with or without HKL. Furthermore, to induce AHR,
mice were also challenged with OVA intranasally (protocol 1, Fig. 1),
following which AHR was measured in a whole body plethysmograph by
challenge with increasing concentrations of methacholine. Fig. 2 demonstrates that immunization of
BALB/c mice with OVA s.c. and intranasally resulted in the development
of significant AHR. OVA-primed mice immunized with OVA plus HKL as an
adjuvant at the time of the first boost showed dramatically reduced
AHR, to levels as low as in control mice, indicating that HKL as an
adjuvant inhibited the development of AHR in OVA-immunized BALB/c
mice.
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At the day of sacrifice, lung histology was examined after
fixation and staining with hematoxylin and eosin. Lung sections from
BALB/c mice that were immunized with OVA without HKL as an adjuvant
showed significant airway inflammation with peribronchiolar and
perivascular infiltrates, consisting of lymphocytes, eosinophils and
some neutrophils (Fig. 3A). In
contrast, lung sections from mice that were immunized with OVA and HKL
as adjuvant showed almost normal lung histology, with only marginal
perivascular and peribronchiolar lymphocytic infiltrates (Fig. 3B). Thus, HKL as adjuvant during the immunization
significantly reduced airway inflammation in OVA-immunized BALB/c
mice.
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The histopathologic analysis was extended by examination of the
cell numbers and types in the BAL fluid, which was harvested 5 days
after the last intranasal challenge with OVA. The total number of cells
recovered in the BAL fluid of BALB/c mice boosted with OVA plus HKL as
an adjuvant was significantly lower than that in the control group
(Fig. 4). Furthermore, vaccination with
HKL as an adjuvant in the immunization protocol significantly reduced
the proportion of eosinophils from 48% in the control group to 11% in
HKL-treated mice (Fig. 4). These results demonstrate that HKL as an
adjuvant significantly reduced the total cell number and the proportion
of eosinophils recovered in the BAL fluid of OVA-immunized mice and
confirm the results observed with lung histology.
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To determine whether the reduced AHR in mice immunized with HKL as
adjuvant correlated with alteration of cytokine profiles in
CD4+ T cells, mice were sacrificed 4 days after
measurement of AHR. Draining LN were removed and LN cells were
stimulated with OVA in vitro. Fig. 5
shows that cells from mice immunized with OVA s.c. and intranasally
produced high levels of IL-4 and low levels of IFN-. In contrast,
immunization with OVA plus HKL as adjuvant inhibited IL-4 production
and greatly enhanced IFN- synthesis.
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shows that
vaccination with OVA plus HKL adjuvant greatly reduced anti-OVA IgE
Ab responses and enhanced anti-OVA IgG2a Ab responses as compared
with control mice immunized with OVA alone. Levels of anti-OVA IgG1
Ab were not significantly different in the two groups (711 ng/ml
anti-OVA IgG1 in the group vaccinated with OVA plus HKL adjuvant vs
823 ng/ml IgG1 anti-OVA in the OVA/IFA control group). HKL must be in close physical association with OVA to inhibit responses
To determine whether HKL had a generalized effect on immune
responses or affected only responses to Ags in close physical
association with it, mice were immunized with HKL and OVA in separate
footpads. Fig. 6 shows that mice which
received HKL and OVA together in the same footpad showed greatly
reduced AHR, whereas mice which received the HKL in a different footpad
from the OVA showed only minimal reduction in AHR. When HKL and OVA
were injected separately in different footpads, the reduction of IL-4
and the increase of IFN- were about 50% of that when administered
together (data not shown). Thus, the inhibition of AHR in OVA-primed
mice was most efficient when HKL and the Ag were in close physical
association with each other.
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To determine whether HKL as an adjuvant could reverse established
AHR in addition to inhibiting the development of AHR, mice were boosted
with OVA plus HKL on day 39, after the establishment of AHR (protocol
2, Fig. 1). Fig. 7A shows that
AHR was present before administration of HKL, but 10 days after
administration of HKL with OVA there was a significant reduction in
AHR. This protective effect with HKL could not be detected 3 days after
the mice had received HKL, indicating that it required at least 10 days
after administration of HKL to develop. Control mice that received OVA
without HKL showed high AHR at all time points.
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production, and decreased OVA-specific IL4 and IgE production
(Fig. 7B). These results demonstrate that HKL as
an adjuvant not only prevents the development of AHR when given during
the earlier phase of the immunization protocol but also reverses
established AHR and the cytokine profiles of CD4+
T cells. The effect of HKL on the development of AHR is mediated by CD8+ T cells
To investigate the mechanism by which HKL affected OVA-specific
responses, we administered blocking Ab to IL-12 or depleting Ab to
CD8+ T cells during the immunization protocol. As
expected, mice immunized with OVA had high airway reactivity, which was
reduced by vaccination with OVA plus HKL in the presence of a control
mAb (Fig. 8A). However,
treatment with anti-CD8 mAb reversed the effect of HKL as adjuvant
and restored OVA-induced AHR. Treatment with anti-IL-12 mAb partly
eliminated the effect of HKL on AHR in OVA-immunized mice. In addition,
the reduction by HKL of OVA-specific IgE levels was partially reversed
by treatment with either anti-CD8 or anti-IL12 mAb (Fig. 8B). Furthermore, treatment of mice with anti-CD8 mAb
partially reversed the effect of HKL on IFN- and IL-4 production.
These results indicate that both CD8+ T cells and
IL-12 play a role in the immunomodulatory effects of HKL on the
AHR.
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Because HKL as adjuvant strongly induced IFN- production (Fig. 4), we asked whether HKL also increased IL-18 production. We analyzed
IL-18 mRNA levels by semiquantitative PCR analysis in splenic adherent
cells cultured in vitro with HKL. Fig. 9A demonstrates that HKL
induced a significant quantity of IL-18 mRNA expression after 8, 16,
and 24 h of culture. Moreover, treatment of mice in vivo with OVA
with HKL, but not with OVA alone, induced IL-18 mRNA expression in
draining LN (Fig. 9B). The mRNA expression was detected
24 h but not at 12 h after immunization. These data indicate
that the induction of IL-18 expression is associated with the
immunomodulatory effects of HKL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Asthma is characterized by the overproduction of the Th2 cytokines
IL-4, IL-5, and IL-13, which initiate and sustain the allergic
asthmatic inflammatory response by enhancing the production of IgE and
the growth, differentiation, and recruitment of mast cells, basophils,
and eosinophils (1). The Th2-driven inflammatory process
may be a consequence of a relative insufficiency in IFN- production
because IFN- can inhibit the development of Th2 responses
(22). In addition, clinical studies demonstrated that
reduced IFN- secretion in neonates is associated with the subsequent
development of atopy (23). Furthermore, a predisposition
toward the overproduction of Th1 cytokines may protect against atopy,
because patients with multiple sclerosis, rheumatoid arthritis, or
infection with tuberculosis (conditions associated with increased
production of Th1 cytokines) have a reduced predisposition toward the
development of atopy (24, 25, 26, 27). These studies together
suggest that methods to enhance IFN- production might be clinically
useful in the treatment of allergic asthma.
Indeed, immunotherapies and immune modulatory approaches that enhance
Th1-dominated responses appear to be beneficial for allergic
individuals (8, 28), and in animal models of allergic
disease (10, 12, 29, 30, 31, 32, 33). Immunotherapies in these models,
however, while effective in preventing the development of AHR, have not
been shown to reverse established AHR. IL-12 administered
intratracheally to mice has been shown to reverse Ag-induced AHR and
inflammation in one study, but the effect of IL-12 has been
controversial (29, 34, 35). In contrast, our current
report describes an immunotherapy that is highly effective in reversing
ongoing AHR. This reversal of AHR with HKL as adjuvant was associated
with a significant increase in IFN- production and a significant
reduction of IL-4 production, in allergen-specific IgE production, and
in IL-10 production (13). The reversal in AHR with HKL as
an adjuvant required only one dose of the HKL plus Ag, and suggests
that immunotherapy with HKL might be effective in patients with asthma,
who by definition have ongoing AHR.
The potent capacity of HKL to reverse established AHR and inflammation
might be due to the fact that HKL activates multiple immunological
mechanisms. Listeria monocytogenes is a Gram-positive,
facultative intracellular bacterium, which elicits a strong classical
cell-mediated immune response, characterized by the presence of potent
Ag-specific CD8+ killer cells (36, 37). The L. monocytogenes proteins listeriolysin
O and p60 are processed through the MHC class I pathway and
stimulate protective CD8+ CTL responses
(38, 39). In our model system, CD8+
T cells induced by HKL plus Ag may play an important role in down
modulating AHR, because treatment with anti-CD8 mAb reversed the
inhibitory effect of HKL on AHR. The Listeria cell wall
component lipoteichoic acid potently induces IL-12 production in
macrophages (40, 41). The effect of IL-12 is thought to be
due to the fact that it stimulates the production of IFN- by NK
cells and T cells, which further enhances Th1
CD4+ T cell development, activates microbicidal
activity of macrophages, and promotes the development of cell-mediated
immune responses (42, 43). Moreover, our study
demonstrated that HKL not only induces the production of IL-12 but also
stimulates transcription of IL-18 mRNA and presumably production of
IL-18 protein. IL-18 is a product of activated macrophages and Kupffer
cells and is 10-fold more potent than IL-12 in driving the development
of Th1 cytokine synthesis in naïve and memory T cells
(44). IL-18 synergizes with IL-12 in inducing IFN-
production (43), in inhibiting IgE production in B cells
(45), and in promoting the differentiation of
CD8+ T cells (46) and possibly
CD8+ 
cells, which have been shown to
inhibit AHR (47). Production of both IL-12 and IL-18 may
be enhanced by the presence of CpG ISS motifs in Listeria
DNA, which can induce IL-12 and IL-18 (48). However, we do
not believe that the activity of Listeria as an adjuvant is
due only to CpG ISS, as may be the case with killed mycobacteria
(49), which appears to be less effective than HKL in
inhibiting Th2-dominated immune responses (13, 32).
Finally, recent studies in our laboratory suggest that heat killed
Listeria also induces TGF-ß production in vivo, which may
provide an additional mechanism that may down-regulate allergic
inflammation and AHR (our unpublished observations). Thus, the
effectiveness of HKL as an adjuvant may therefore depend on the
induction of IL-12, IL-18, and IFN- production, and well as the
induction of CD8 and Th1 cells.
The mechanism by which HKL as an adjuvant reverses established AHR and inflammation may also involve the conversion of OVA-specific CD4+ Th2 cells into Th1 cells, or the inhibition or attrition of Th2 effector cells over time while a protective immune response develops from uncommitted OVA-specific precursor cells. Because the cytokine profile of Th2 effector cells are relatively fixed, we speculate that attrition of Th2 effector cells as well as the induction of several types of Th2-inhibiting regulatory cells are involved in this process. We recently showed that localization of Ag-specific Th1 cells in the lungs caused airway inflammation and lung injury, and did not reduce AHR (50), suggesting that other types of cells in addition to Th1 cells (e.g., CD8 cells or CD4 Th3 cells) (51) are induced by HKL. The fact that CD8 cells and TGF-ß production are induced by HKL supports this idea. The generation of such OVA-specific regulatory cells by HKL may enhance the development of a protective immune response, such that further exposure to allergen does not result in AHR and airway inflammation. The existence of such regulatory cells and the mechanisms by which they affect allergic inflammatory processes, and how long the effect of HKL persists are currently being investigated.
The attractiveness of Listeria as adjuvant therapy lies also
in the fact that its immunomodulatory effects remain largely Ag
specific. Listeria had minimal effect on AHR, IgE, and
cytokine production unless the Listeria was administered in
a mixture with the Ag. Thus Listeria did not induce a
generalized enhancement of IFN- production in recipients, but rather
induced a protective response that was Ag specific. The capacity to
induce Ag-specific modulation is very important, because this
specificity avoids nonspecific immune augmentation, which could result
in the development of autoimmune diseases. Ag-specific therapy is
feasible for the treatment of allergic rhinitis and allergic asthma,
because the major offending allergens are virtually always identified.
Thus, the use of Listeria as an adjuvant could greatly
improve and refine conventional allergen immunotherapy
(9), which currently requires multiple injections of
soluble allergen over several years time and is associated with
frequent failures. The safety issues of using Listeria in
humans may not be of major concern because killed rather than live
Listeria is effective for immune modulation. In addition,
live Listeria is a relatively common organism, and is a
pathogen primarily in immunosuppressed patients or in the setting of
pregnancy.
In conclusion, we demonstrated that allergen immunotherapy with HKL as adjuvant greatly inhibited the development of AHR and airway inflammation. Immunotherapy with HKL as adjuvant reversed ongoing airway disease, and converted allergic inflammatory responses into protective immune responses. The effect involved multiple mechanisms, including the induction of IL-18 and CD8+ T cells, activation of the innate immune system and inhibition of Th2 cytokine production. Our results suggest that allergen immunotherapies with HKL as adjuvant for asthma may be feasible, and further studies in humans with HKL are warranted to determine the effectiveness of HKL in human disease.
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Acknowledgments |
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Footnotes |
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2 G.H. and P.Y. contributed equally to the completion of these studies.
3 Current address: Martin-Luther-University, Department of Pediatrics and Biocenter, 06120 Halle/Saale, Germany.
4 Address correspondence and reprint requests to Dr. Dale T. Umetsu, Department of Pediatrics, Stanford University, Room H307, Stanford, CA 94305-5208. E-mail address:
5 Abbreviations used in this paper: AHR, airway hyperreactivity; HKL, heat-killed Listeria monocytogenes; LN, lymph node; BAL, bronchoalveolar lavage.
Received for publication August 2, 1999. Accepted for publication October 15, 1999.
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  M. Maret, C. Ruffie, S. Letuve, A. Phelep, O. Thibaudeau, J. Marchal, M. Pretolani, and A. Druilhe A Role for Bid in Eosinophil Apoptosis and in Allergic Airway Reaction J. Immunol., May 1, 2009; 182(9): 5740 - 5747. [Abstract] [Full Text] [PDF]
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 M. Maret, C. Ruffie, B. Periquet, A.-M. Campo, M. Menevret, A. Phelep, K. Dziewiszek, A. Druilhe, and M. Pretolani Liposomal Retinoic Acids Modulate Asthma Manifestations in Mice J. Nutr., December 1, 2007; 137(12): 2730 - 2736. [Abstract] [Full Text] [PDF]
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 J. E. Korf, G. Pynaert, K. Tournoy, T. Boonefaes, A. Van Oosterhout, D. Ginneberge, A. Haegeman, J. A. Verschoor, P. De Baetselier, and J. Grooten Macrophage Reprogramming by Mycolic Acid Promotes a Tolerogenic Response in Experimental Asthma Am. J. Respir. Crit. Care Med., July 15, 2006; 174(2): 152 - 160. [Abstract] [Full Text] [PDF]
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 A. J. M. van Oosterhout and N. Bloksma Regulatory T-lymphocytes in asthma Eur. Respir. J., November 1, 2005; 26(5): 918 - 932. [Abstract] [Full Text] [PDF]
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A. J. M. van Oosterhout and A. C. Motta Th1/Th2 paradigm: not seeing the forest for the trees? Eur. Respir. J., April 1, 2005; 25(4): 591 - 593. [Full Text] [PDF]
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 J.-K. Peng, J.-S. Lin, J. T. Kung, F. D. Finkelman, and B. A. Wu-Hsieh The combined effect of IL-4 and IL-10 suppresses the generation of, but does not change the polarity of, type-1 T cells in Histoplasma infection Int. Immunol., February 1, 2005; 17(2): 193 - 205. [Abstract] [Full Text] [PDF]
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 H. Kuipers, C. Heirman, D. Hijdra, F. Muskens, M. Willart, S. van Meirvenne, K. Thielemans, H. C. Hoogsteden, and B. N. Lambrecht Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization J. Leukoc. Biol., November 1, 2004; 76(5): 1028 - 1038. [Abstract] [Full Text] [PDF]
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V. Smart, P. S. Foster, M. E. Rothenberg, T. J. V. Higgins, and S. P. Hogan A Plant-Based Allergy Vaccine Suppresses Experimental Asthma Via an IFN-{gamma} and CD4+CD45RBlow T Cell-Dependent Mechanism J. Immunol., August 15, 2003; 171(4): 2116 - 2126. [Abstract] [Full Text] [PDF]
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G. Wohlleben, J. Muller, U. Tatsch, C. Hambrecht, U. Herz, H. Renz, E. Schmitt, H. Moll, and K. J. Erb Influenza A Virus Infection Inhibits the Efficient Recruitment of Th2 Cells into the Airways and the Development of Airway Eosinophilia J. Immunol., May 1, 2003; 170(9): 4601 - 4611. [Abstract] [Full Text] [PDF]
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X.-M. Li, K. Srivastava, J. W. Huleatt, K. Bottomly, A. W. Burks, and H. A. Sampson Engineered Recombinant Peanut Protein and Heat-Killed Listeria monocytogenes Coadministration Protects Against Peanut-Induced Anaphylaxis in a Murine Model J. Immunol., March 15, 2003; 170(6): 3289 - 3295. [Abstract] [Full Text] [PDF]
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D. M. Walter, J. J. McIntire, G. Berry, A. N. J. McKenzie, D. D. Donaldson, R. H. DeKruyff, and D. T. Umetsu Critical Role for IL-13 in the Development of Allergen-Induced Airway Hyperreactivity J. Immunol., October 15, 2001; 167(8): 4668 - 4675. [Abstract] [Full Text] [PDF]
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D. M. Walter, C. P. Wong, R. H. DeKruyff, G. J. Berry, S. Levy, and D. T. Umetsu IL-18 Gene Transfer by Adenovirus Prevents the Development of and Reverses Established Allergen-Induced Airway Hyperreactivity J. Immunol., May 15, 2001; 166(10): 6392 - 6398. [Abstract] [Full Text] [PDF]
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 D. Mizuki, T. Miura, S. Sasaki, M. Mizuki, H. Madarame, and A. Nakane Interference between Host Resistance to Listeria monocytogenes Infection and Ovalbumin-Induced Allergic Responses in Mice Infect. Immun., March 1, 2001; 69(3): 1883 - 1888. [Abstract] [Full Text] [PDF]
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