| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Medicine,
Molecular Virology, and
Pediatrics, University of Wisconsin, Madison, WI 53792
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. RV16
induced a significant shift from CD18dim to
CD18bright, but did not affect EOS expression of CD54,
CD69, or HLA-DR. Finally, RV16 did not induce superoxide production
from peripheral blood EOS. These findings suggest that RV16 also binds
to airway EOS, which resemble granulocyte-macrophage CSF-treated blood
EOS in terms of high expression of ICAM-1. Furthermore, our findings
suggest that EOS could participate in RV-induced immune responses
through Ag presentation and T cell activation. By activating
RV-specific T cells, EOS may play an important role in the initiation
of antiviral T cell responses, and these effects could also contribute
to enhanced airway inflammation and increased asthma symptoms in
susceptible individuals. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To define interactions between RV and EOS, we performed the following experiments to identify binding of RV16 to EOS, and to measure the effects of RV16 on expression of cell surface activation markers and superoxide production. In addition, we have explored the possibility that EOS could also play an indirect role in RV-induced inflammation by presenting viral Ags to and thus activating RV-specific T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
EOS were isolated from peripheral blood of subjects ages 22 to 54 yr with either allergic rhinitis (n = 13) or atopic asthma (n = 8), including four subjects with both disorders. All were skin-prick positive to at least one environmental allergen, most commonly house-dust mite, ragweed pollen, or cat dander. Inhaled ß2-agonists or corticosteroids were taken as needed, but none of the subjects was taking systemic medications. The study protocol was approved by University of Wisconsin Human Subjects Committee (Madison, WI), and informed consent was obtained from all subjects before entry into the study.
mAb and reagents
Mouse anti-human CD54, CD69, HLA-DR, and mouse IgG1 labeled
with either phycoerythrin or fluorescein were obtained from Becton
Dickinson (San Jose, CA). Anti-CD16-coated magnetic microbeads and
steel-meshed columns, type c, were purchased from Miltenyi-Biotech
(Auburn, CA). A blocking anti-ICAM-1 mAb (C78.4A) was kindly
provided by Dr. J. Greve (Miles Pharmaceutical Division, West Haven,
CT). A pair of anti-IFN- mAb used for ELISA was purchased from
Endogen (Cambridge, MA). PMA, FMLP, superoxide dismutase, and horse
heart ferricytochrome c (type VI) were obtained from Sigma
Chemical Co. (St. Louis, MO).
Rhinovirus suspensions
RV16 was kindly supplied by Dr. Elliot Dick (Department of Preventive Medicine, University of Wisconsin). For experiments in which the effects of RV16 on EOS activation were measured, the virus was purified by centrifugation through a sucrose density gradient, as previously described (12), to remove soluble factors of HeLa cell origin. Radiolabeled ([35S]methionine) RV16 was prepared as previously described (12), and was used to measure binding of RV16 to EOS. Viral infectivity was expressed in tissue culture infective units50 (TCID50), and all virus stocks were stored at -70°C until needed.
Cell separation
EOS were purified from peripheral blood using the method developed by Hansel (13), with modifications. Briefly, heparinized whole blood was centrifuged (700 x g, 20 min) over a Percoll gradient (density 1.090 g/ml; Pharmacia Biotech, Piscataway, NJ) to separate mononuclear cells from granulocytes. After removal of the mononuclear cell band, RBC were lysed by twice incubating (30 s) with sterile deionized water. The remaining white blood cells were incubated with anti-CD16-coated microbeads (100 µl/2 x 108 cells) for 40 min and were then passed through steel mesh columns that had been previously washed with 2% newborn calf serum. The cells in the eluent were stained (Diff Quik; Baxter, Miami, FL), and 400 cells were examined microscopically; greater than 99% of the cells were EOS, and the few remaining cells were either neutrophils or mononuclear cells.
Binding of 35S-RV16 to EOS
EOS (3–4 x 106) suspended in 100 µl PBS (pH 7.4), with 0.1% BSA, 0.01% CaCl2, and 0.01% MgCl2, were incubated (room temperature, 60 min) in 5-ml polypropylene tubes on a rocking platform with 35S-RV16 (4000 viral particles/cell). Some EOS samples were preincubated (overnight, 37°C) with GM-CSF (100 pM) to increase expression of ICAM-1, the cellular receptor for major group RV such as RV16. To block RV16/EOS binding, additional samples were incubated with either mAb specific for ICAM-1 (20 µg/ml), or an equivalent amount of an isotype control IgG1. After the initial incubation, the cells were washed twice in PBS and the supernatants were discarded. Cell pellets were then suspended in 4 ml of scintillation fluid, and cell-associated radioactivity (cpm) was measured in a standard scintillation counter. All samples were run in duplicate.
Stimulation of RV-specific T cell clones by EOS
RV16-specific CD4+ T cell clones were prepared from the peripheral blood of a healthy RV16-seropositive donor using Ag stimulation and limiting dilution (14). T cell clones (1 x 104/well) were incubated (48 h, 37°C, 5% CO2) in round-bottom 96-well plates with either RV16 (10 TCID50/APC) or control fluid (CF, medium from uninfected HeLa cells) in the presence of either 1 x 105 autologous irradiated (5000 rad) PBMC, 1 x 105 autologous EOS, 1 x 105 allogeneic EOS, or no APC. The cells were then incubated for an additional 18 h with tritiated thymidine (1 µCi/well), and thymidine uptake was quantitated in a scintillation counter. Results are expressed as stimulation indices (SI), calculated by dividing cpm of stimulated samples by cpm of cells incubated with medium alone.
IFN- ELISA
Cell-free supernatants from cultured EOS were stored at -80°C
for determination of IFN- levels by a two-step sandwich ELISA
assay, as previously described (15). The sensitivity of the assay was
12 pg/ml, and coefficient of variation <10%.
Flow cytometry
EOS (2 x 106/ml) were cultured overnight
in RPMI with 10% FCS, glutamine, and penicillin/streptomycin, in the
presence or absence of purified RV16 (10 TCID50/cell).
Cells incubated with GM-CSF (100 pM) and TNF-
(100 ng/ml) were used
as positive controls for the induction of cell surface markers. For
some experiments, EOS were incubated with GM-CSF (100 pM), TNF- (100
ng/ml), or both of these cytokines to increase ICAM-1 expression; the
cells were then washed, and incubated (37°C, 5% CO2) for
an additional 24 h with RV16 (10 TCID50/cell). The
following morning, aliquots of EOS (1–2 x 105
cells/100 µl sample) were washed in PBS and incubated (30 min on ice)
with 5 µl of phycoerythrin-conjugated mAbs specific for CD54, HLA-DR,
CD69, or phycoerythrin-conjugated mouse IgG2a (isotype control), and
expression of these surface proteins was analyzed using flow cytometry,
as previously described (15). Measurements of CD54, HLA-DR, and CD69
expression were expressed as median fluorescent units. CD18 expression,
which had a bimodal distribution, was expressed in terms of percentage
of CD18bright or percentage of CD18dim
cells.
Superoxide anion (O2-) generation
Assessment of O2- generation was
performed by superoxide dismutase-inhibitable cytochrome c
reduction as previously described (16). EOS were either freshly
isolated, or for some experiments, incubated overnight with GM-CSF (100
pM) ± TNF- (100 ng/ml) to increase the expression of ICAM-1 and
enhance binding of RV16. The EOS (1 x 105 cells/well)
were then suspended in HBSS/0.1% gelatin and added to 96-well plates
along with 100 nM cytochrome c. Controls included cells
activated with PMA (1 ng/ml), a strong inducer of superoxide
(O2-) production, and cells incubated in
buffer alone to measure spontaneous release. Duplicate samples of EOS
were incubated (37°C) with either purified RV16 (10
TCID50/cell), an equivalent amount of CF, or medium alone,
and absorbance (550 nM) was measured for 2 h.
O2- generation was calculated using an
extinction coefficient of 21.1 x
10-3M-1cm-1, and results were
calculated as nanomols of reduced cytochrome c/5 x
105 cells, minus spontaneous O2-
generation.
Statistical analysis
Data were analyzed using SYSTAT software (version 5; Systat,
Evanston, IL). Analysis of variance was performed after normalization
of the data on O2- generation and flow
cytometry. Paired data were evaluated with Fisher’s least significant
test and the paired Student’s t test, and a p
value of 
0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether RV binds to EOS, 35S-RV16
was incubated with EOS, and cell-associated radioactivity was measured.
After a 60-min incubation with radiolabeled RV16, there was little
radioactivity associated with unstimulated EOS, and this binding was
not sensitive to anti-ICAM-1 mAb (Fig. 1
A). In contrast, EOS
pretreated with GM-CSF, which induces ICAM-1 expression on EOS (17),
bound more 35S-RV16, and RV binding was inhibited by
anti-ICAM-1 (Fig. 1B). These findings demonstrate
that RV16, a major group RV, binds to EOS, and that GM-CSF increases
this binding, probably by increasing expression of ICAM-1.
|
To determine whether EOS can participate in RV-induced immune
responses by serving as APC, we measured virus-induced proliferation of
RV16-specific T cell clones in the presence of either autologous iPBMC,
autologous EOS, allogeneic EOS, or no APC. Of the nine RV16-specific T
cell clones tested, eight proliferated vigorously (SI = 15) in the
presence of RV16 and autologous EOS (Fig. 2A). This degree of T
cell stimulation was similar to that induced by RV16 plus autologous
iPBMC (SI = 12, Fig. 2B). No such proliferation
occurred when RV16 was added along with allogeneic EOS (Fig. 2C), or in the presence of virus alone without APC
(Fig. 2D). Background thymidine incorporation was
slightly higher when the T cells were incubated with iPBMC compared
with EOS (1486 vs 270, median cpm, p = 0.02,
n = 8), suggesting that PBMC produce a greater degree
of nonspecific stimulation in the absence of RV16. Proliferation of T
cell clones was greatest at higher RV concentrations (1–10
TCID50/cell), and proliferative responses to lower doses of
Ag were similar, regardless of whether PBMC or EOS were used as APC
(data not shown).
|
). T cell proliferation
declined when fewer PBMC/well were used, and only a slight degree of
proliferation occurred (157 cpm) with 104 PBMC/well. These
findings rule out the possibility that the small number of mononuclear
cells in the EOS preparations made a significant contribution to T cell
proliferation mediated by the EOS.
|
by T cells stimulated by Ag and EOS
RV16-specific T cell clones were incubated with RV16 or CF alone
in the presence of either autologous EOS, iPBMC, or medium alone for
24 h, and supernatants were collected and assayed for IFN-
(Table I). T cell clones incubated with
RV16 in the presence of either PBMC or EOS secreted large amounts of
IFN-. In contrast, RV16 induced very little IFN- from either EOS
or PBMC in the absence of T cell clones, or from clones in the absence
of APC. Both EOS and PBMC caused T cell clones to secrete low levels of
IFN- in the absence of Ag, and this nonspecific IFN- secretion
was greater in the presence of PBMC.
|
EOS that are functionally up-regulated express increased amounts
of several cell surface markers, including ICAM-1 (CD54), CD69, HLA-DR,
and CD18 (18). We incubated blood EOS with RV16 (10
TCID50/cell), and then measured effects on these
surface proteins using flow cytometry. RV16 did not affect the surface
expression of ICAM-1, CD69, or HLA-DR (Fig. 4). Furthermore, although preincubation
with GM-CSF (100 pM), TNF- (100 ng/ml), or the combination of these
cytokines induced increased expression of these cell surface markers,
sequential incubation (24 h, 37°C) with these cytokines followed by
RV16 did not cause additional increases (Table II). In fact, RV16 reduced ICAM-1
detection by about 50% on cells preincubated with cytokine(s),
probably by competing for the same binding site as the detection
Ab.
|
|
), and 17% of the
cells were CD18dim (mean, n = 3).
Incubation with RV16 shifted the distribution of CD18 so that only 5%
of cells were CD18dim (mean, n = 3,
p < 0.01), and cytokine (TNF- and GM-CSF)
incubation had similar effects.
|
To determine whether RV binding to EOS caused cell activation,
peripheral blood EOS were incubated overnight with GM-CSF to increase
ICAM-1 expression and enhance RV16 binding, and then EOS
O2- production was evaluated at 0 to 120
min after incubation with RV16 (10 TCID50/cell), PMA (1
ng/ml), or CF. In contrast to the vigorous superoxide production
induced by PMA, RV16 had no effect on O2-
generation by EOS (Fig. 6). Additional
experiments performed with freshly isolated EOS, or EOS treated with
both GM-CSF and TNF- to maximally up-regulate ICAM-1 expression,
yielded similar results (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In addition to binding RV, EOS also presented viral Ag to RV16-specific
T cells, inducing T cell proliferation and IFN- secretion. T cell
proliferation was mediated by autologous, but not allogeneic EOS,
proving that this was an MHC-restricted process. Although there were
small numbers (<1%) of mononuclear cells and neutrophils present in
the EOS preparations, titration of iPBMC as APC demonstrated that these
few cells could not have accounted for the observed T cell
proliferation. Although there are previous reports that EOS can present
purified protein Ags (20, 21), allergens (17), or superantigens (22) to
specific T cells, this is the first report that EOS may participate in
virus-induced immune responses by processing and presenting complex
viral proteins and/or intact viral particles.
Our data indicate that EOS have the potential to activate RV-specific T
cells, and this could have several effects on airway physiology. First,
activation of virus-specific T cells is likely to contribute to
antiviral responses, leading to clearance of virus and resolution of
symptoms. On the other hand, virus-specific T cell responses could also
augment pre-existing airway inflammation, and thereby potentiate
respiratory symptoms (23). In particular, virus-induced IFN- could
increase adhesion molecule expression, leading to increased recruitment
of cells to the airway, and through the induction of ICAM-1 (24, 25),
IFN- could also potentiate RV binding to epithelial cells and airway
inflammatory cells.
While we found little evidence that RV directly activates EOS, it is important to consider that the process of Ag presentation is associated with bidirectional cellular signals and activation (26), and that EOS could be activated during the process of Ag presentation. Specifically, the engagement of cell surface receptors during T cell/EOS binding, along with the secretion of cytokines by the activated T cell, could augment EOS inflammatory functions. In fact, it has been demonstrated that activated T cells bind to either resting or PMA-treated EOS via VLA-4 and CD18, and that this binding causes EOS activation, as indicated by increased expression of CD11b and CD66b surface markers (27). Furthermore, we found that RV incubation enhanced EOS CD18 expression, which could lead to greater T cell/EOS binding. When considered together, these findings suggest that RV could initiate a feedback loop between EOS and virus-specific T cells that could potentially lead to a bidirectional activation of both cell types.
The presence of increased numbers of EOS in the airway, as occurs in asthma, may counteract the suppressive effects of alveolar macrophages, which are not effective APC, and can suppress activation of T cells under some conditions (28). Enhancement of T cell proliferation and cytokine secretion in asthma could cause increased inflammation, and ultimately, produce greater airway obstruction, bronchial hyper-responsiveness, and clinical symptoms (23). Thus, the availability of increased numbers of EOS in such subjects to interact with RV and activate virus-specific T cells could, in part, explain the more severe clinical manifestations of RV infection that occur in asthmatic vs normal individuals.
Our data suggest that RV produces small changes in EOS phenotype, as
indicated by a significant shift from CD18dim to
CD18bright expression, but major changes in cell surface
markers were not observed. Likewise, RV did not trigger superoxide
release from EOS, even after priming with cytokines (GM-CSF ±
TNF-) to enhance ICAM-1 expression. Thus, these observations suggest
that the EOS activation observed during natural RV infection may not be
due to direct effects of the virus on EOS. These results need to be
tempered by the fact that there are functional and phenotypic
differences between primed peripheral blood EOS and airway EOS (29),
and additional experiments to examine the effects of RV on airway EOS
are now in progress in our laboratory.
There are several other events in the airway biology during RV infection that are more likely to influence EOS function. For example, RV infection induces RANTES secretion in vivo (30), and inoculation of epithelial cells with RV induces secretion of cytokines such as GM-CSF in vitro (31); both of these cytokines have profound effects on EOS recruitment and function. RANTES is a chemoattractant for EOS and memory T cells, both of which are regarded as key cells in asthmatic airway inflammation (32, 33). Furthermore, GM-CSF is a potent activator of EOS survival and adhesion molecule expression, and is a cofactor for EOS superoxide production and degranulation (18, 34, 35). These limited data suggest that EOS activation during RV infections is more likely to occur as a consequence of T cell or epithelial cell activation than as a result of direct activation of EOS by RV.
In conclusion, to explore features of the immune response to RV infections that may be unique to patients with allergies and/or asthma, we have examined effects of RV on EOS phenotype and function in vitro. Our findings suggest that EOS could contribute to RV-induced immune responses by binding viral particles and functioning as APC. EOS-mediated T cell activation could initiate both antiviral responses and proinflammatory effects that could augment pre-existing airway inflammation in diseases such as asthma. We found little evidence that RV directly potentiates EOS inflammatory functions; rather, it is likely that EOS activation in RV infections in vivo is mediated through RV actions on activated airway T cells, epithelial cells, or other inflammatory cells, leading to the generation of cytokines and other mediators that promote EOS inflammatory function.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Kaplan Hospital, 76100 Rehovot, Israel.
3 Address correspondence and reprint requests to Dr. James E. Gern, H4/438 CSC, University of Wisconsin Hospital, 600 Highland Avenue, Madison, WI 53792-4108. E-mail address:
4 Abbreviations used in this paper: RV, rhinovirus; CF, control fluid; EOS, eosinophil; GM-CSF, granulocyte-macrophage CSF; iPBMC, irradiated PBMC; 35S-RV16, 35S-labeled rhinovirus type 16; SI, stimulation index; TCID50, 50% tissue culture-infective dose.
Received for publication December 2, 1996. Accepted for publication October 8, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137:245.[Abstract]
are increased in the nasal aspirates of children with virus-associated asthma. Am. J. Respir. Crit. Care Med. 151:A385. (Abstr.).
This article has been cited by other articles:
|
|
 |
|
  U. M. Padigel, J. J. Lee, T. J. Nolan, G. A. Schad, and D. Abraham Eosinophils Can Function as Antigen-Presenting Cells To Induce Primary and Secondary Immune Responses to Strongyloides stercoralis. Infect. Immun., June 1, 2006; 74(6): 3232 - 3238. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-Z. Shi Eosinophils function as antigen-presenting cells J. Leukoc. Biol., September 1, 2004; 76(3): 520 - 527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Message and S. L. Johnston Host defense function of the airway epithelium in health and disease: clinical background J. Leukoc. Biol., January 1, 2004; 75(1): 5 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. de Kluijver, C. E. Evertse, J. K. Sont, J. A. Schrumpf, C. J. G. van Zeijl-van der Ham, C. R. Dick, K. F. Rabe, P. S. Hiemstra, and P. J. Sterk Are Rhinovirus-induced Airway Responses in Asthma Aggravated by Chronic Allergen Exposure? Am. J. Respir. Crit. Care Med., November 15, 2003; 168(10): 1174 - 1180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Nagase, S. Okugawa, Y. Ota, M. Yamaguchi, H. Tomizawa, K. Matsushima, K. Ohta, K. Yamamoto, and K. Hirai Expression and Function of Toll-Like Receptors in Eosinophils: Activation by Toll-Like Receptor 7 Ligand J. Immunol., October 15, 2003; 171(8): 3977 - 3982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Adamko, A. D. Fryer, B. S. Bochner, and D. B. Jacoby CD8+ T Lymphocytes in Viral Hyperreactivity and M2 Muscarinic Receptor Dysfunction Am. J. Respir. Crit. Care Med., February 15, 2003; 167(4): 550 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Adamko, B. L. Yost, G. J. Gleich, A. D. Fryer, and D. B. Jacoby Ovalbumin Sensitization Changes the Inflammatory Response to Subsequent Parainfluenza Infection: Eosinophils Mediate Airway Hyperresponsiveness, M2 Muscarinic Receptor Dysfunction, and Antiviral Effects J. Exp. Med., November 15, 1999; 190(10): 1465 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Giembycz and M. A. Lindsay Pharmacology of the Eosinophil Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Gern and W. W. Busse Association of Rhinovirus Infections with Asthma Clin. Microbiol. Rev., January 1, 1999; 12(1): 9 - 18. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
5%,
p < 0.01).