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Cutting Edge |
1
* Department of Immunology, Weizmann Institute of Science, and
Sourasky Medical Center, Tel-Aviv, Israel
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Abstract |
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inhibit homing of B cells to the secondary lymphoid organs. In
this study, we demonstrate that similarly low doses of IFN-
down-regulate integrin-mediated adhesion and migration of naive T and
Th2 cells, and have a profound effect on the in vivo homing of naive T
cells to the lymph nodes. Moreover, we show that these low doses of
IFN- have anti-inflammatory effects in an in vivo asthma model.
Thus, in contrast to the proinflammatory effects of IFN- at
relatively high concentrations, low dose IFN- appears to exert
global suppressory effects on T cell trafficking and may have clinical
application as an anti-inflammatory agent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive T lymphocytes traffic through the T cell areas of secondary lymphoid organs in search of Ag presented by dendritic cells (5, 6). Upon Ag recognition, specific T cells proliferate and, in the presence of polarizing cytokines, differentiate into Th1 or Th2 cells, which produce distinct patterns of cytokines and mediate various types of protective and pathological responses (7, 8). After T cell differentiation into Th1 or Th2 phenotypes, the lymph node (LN)4 homing receptors are down-regulated, while expression of tissue homing receptors is acquired (9, 10).
Recently, we demonstrated that to prevent premature encounter with Ag,
immature B cells down-regulate their own integrin-mediated adhesion to
the extracellular matrix (ECM). This inhibition is mediated by IFN-,
which is transcribed and secreted by immature B cells to down-regulate
their migration to the LNs in an autocrine manner. In addition, these
low levels of IFN- were found to dramatically affect homing of
mature B cells to secondary lymphoid organs (11). The
powerful inhibitory effect of IFN- on adhesion of B lymphocytes both
in vitro and in vivo led us to suggest that this cytokine might serve
as a more general modulator of the immune response and have an
influence on other subsets of the immune system. Therefore, in this
study, we analyzed the ability of low levels of IFN- to regulate
migration of T cells in vitro and in vivo. Our findings demonstrate
that low levels of IFN- prevent adhesion and migration of naive T
and Th2 cells in vitro and have anti-inflammatory effects in an in
vivo asthma model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 or BALB/c mice were used at 6–8 wk of age. All animal procedures were approved by the Animal Research Committee at the Weizmann Institute (Rehovot, Israel).
Cells
Spleen cells were obtained from mice as previously described (12). T cells were enriched by using the MACS system (Miltenyi Biotec, Auburn, CA). Spleen cells were incubated with anti-CD45R (B220) magnetic beads, and the CD45 negative cells were collected. CD4+ T cells were enriched using anti-CD4 magnetic beads. To obtain Th1 and Th2 cells, the T cell-enriched population was incubated with Con A (250 µg/ml; Roche, Basel, Switzerland) and IL-12 (3.5 ng/ml; Life Technologies, Rockville, MD) or IL-4 (103 U/ml; Life Technologies) for 96 h. Th1 supernatant was collected from the IL-12-treated cells.
Adhesion assay
Adhesion assays were performed as described previously (11).
Transwell migration
Chemotaxis was assayed by using transwell chambers as was previously described (13).
Tracking of cells in vivo
The assay, based on labeling cells with BCECF-AM (Molecular Probes, Eugene, OR), was performed as described previously (11).
OVA sensitization and challenge
BALB/c mice were immunized i.p. on days 0, 7, and 14 as
previously described (14). The OVA/OVA group was i.p.
injected with 300 µl 0.9% NaCl from day 15 (first day of challenge)
for 5 days, 5 min before each inhalation. The IFN- group was i.p.
injected with murine IFN- (6 U in 300 µl 0.9% NaCl; Genentech,
South San Francisco, CA) from day 15 (first day of challenge) for 5
days, 5 min before each inhalation.
Measurement of airway hyperactivity (AHR)
AHR was determined as previously described (15) by calculating the enhanced expiratory pause (Penn) measured in a plethysmographic box during expiration (Buxco Electronics, Sharon, CT).
bronchoalveolar lavage (BAL)
BAL was performed on day 19, 4 h after the last AHR evaluation as was previously described (14).
Lung histology
Lungs were inflated with 1 ml of 10% formalin until distended. Samples for paraffin sectioning were prepared as described previously (14). Immunohistochemistry was performed as previously described (16). The sections were stained with anti-CD3 (Serotec, Oxford, U.K.) followed by biotinylated anti-rat IgG Ab. The slides were developed using 3,3-diaminobenzidine as a substrate.
Cytoskeleton rearrangement
The cells were analyzed as previously described (13).
Polyclonal stimulation
CD4+ T cells were cultured in the presence
of Con A (2.5 µg/ml; Roche) in the presence or absence of elevated
concentrations of IFN-. Proliferation was determined after 4 days by
incorporation of [3H]thymidine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibit integrin-mediated adhesion and
migration of T cells in vitro
To migrate to LNs or to sites of inflammation, T cells must
interact with components of the ECM (5). To determine
whether low levels of IFN- negatively regulate homing of naive T
cells, we studied their integrin-mediated adhesion to the major ECM
component, fibronectin. 51Cr-labeled naive T
cells were activated with or without PMA, a potent agonist of
integrin-mediated adhesion (17), or stromal-cell-derived
factor 1 (SDF-1), a potent T cell chemoattractant and integrin
agonist (18) in the presence or absence of low levels of
IFN- (0.1 U/ml). Upon activation, T cell adhesion to fibronectin was
dramatically augmented, while in the presence of low levels of IFN-
(Fig. 1
A) the
integrin-mediated adhesion response to PMA or chemoattractant of these
T cells was abolished.
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is secreted at high levels by Th1 (
350 ng/ml,
equivalent to 35,000 U/ml), T cells might encounter high levels of
IFN- in vivo. To investigate whether these high levels of IFN-
have any influence on the homing of naive T cells, we examined the
adhesion of naive T cells to fibronectin in the presence of supernatant
collected from cultured Th1 cells. As can be seen in Fig. 1
A, Th1 cell supernatant did not alter the adhesion response
of PMA- or SDF-1-stimulated T cells, and they were able to increase
their adhesion response in a manner similar to untreated cells.
Moreover, in the presence of high levels of rIFN- (50 u/ml), the
adhesion response was hardly affected (Fig. 1B).
Because Th1 cells secrete 104 times
higher levels of IFN- than used in our assays (19, 20),
our results suggest that while low levels of IFN- regulate adhesion
and migration of cells, high levels of IFN- do not similarly affect
these processes.
We further analyzed the inhibitory effect of low levels of IFN- on
the transwell chemotactic migration of naive T cells. Naive T cells
were suspended with or without low (0.1 U/ml) and higher (1 and 10
U/ml) levels of IFN-, placed in the upper chamber of a transwell,
and were allowed to migrate toward SDF-1 placed in the lower chamber.
Low levels of IFN- inhibited the migration of T cells toward SDF-1
by 30% (Fig. 1C), while higher doses of this cytokine (1
and 10 U/ml) had no inhibitory effect on the migration of the
cells.
In B cells, the inhibitory signal of IFN- is transmitted through the
IFN- receptor, whose engagement leads to inhibition of cytoskeleton
rearrangement (13). To determine whether IFN- similarly
regulates cytoskeletal rearrangement in T cells, we followed actin
polymerization of SDF-1-activated naive T cells, which were
preincubated in the presence or absence of lower (0.1 U/ml) and higher
(1 U/ml) levels of IFN-. As can be seen in Fig. 1D, SDF-1
stimulation induced actin polymerization, which was inhibited by
pretreatment with 0.1 U/ml of IFN-. This inhibition was lost in the
presence of higher levels of this cytokine. Thus, the inhibition of
adhesion and migration of T cells by low levels of IFN- correlate
with reduced actin polymerization.
To directly show that low levels of IFN- are not toxic for the
cells, we analyzed CD4+ T cell proliferation in
the presence or absence of elevated concentrations of IFN-. As can
be seen in Fig. 1E, IFN- did not inhibit the
proliferative response of the cells, showing that the lack of adhesion
and migration of T cells do not result from the cell death.
When naive T cells find their specific Ag, they proliferate and
differentiate into effector and memory T cells. Effector
CD4+ T cells can be subdivided into two
functionally distinct subsets, Th1 and Th2 cells. Because Th2 cells do
not secrete IFN-, we determined whether the migration of Th2 cells
is nevertheless affected similarly by low levels of IFN-. Th2 cells
pretreated with or without low levels of IFN- were placed in the
upper chamber of a transwell and were allowed to migrate toward SDF-1
placed in the lower chamber. As can be seen in Fig. 1F, low
dose IFN- suppressed the migration of SDF-1-activated Th2 cells.
However, Th2 cells had a broader dose response to IFN-, and 1 U/ml
of IFN- inhibited the chemotactic-dependent migration of the cells,
suggesting that naive and effector T cell population respond
differently to various IFN- concentrations.
Low levels of IFN- have an anti-inflammatory function in
vivo
The in vitro powerful inhibitory effect of IFN- on adhesion and
migration of T lymphocytes suggests that this cytokine might regulate
homing of T cells in vivo and serve as an anti-inflammatory
compound. To determine the in vivo effect of IFN- on homing of naive
T cells, we analyzed their entry into the LNs. T cells preincubated in
the presence or absence of IFN- (0.1 or 1 U/ml) were washed, labeled
with the fluorescent dye, and an equal number of IFN-treated and
-untreated cells were injected i.v. into mice. The proportion of
labeled cells recovered in the LNs was determined 3.5 h after
injection. As can be seen in Fig. 1G, compared with the
control (medium-treated) population, homing of IFN--treated cells
into the LNs was almost abolished. Therefore, IFN- inhibits the
migration of naive T cells to the LNs.
To further study the in vivo effect of IFN-, we analyzed its
influence on the effector Th2 cells in an asthma model. Asthma is a
chronic inflammatory disorder of the airways that is characterized by
intermittent episodes of airway obstruction and wheezing. Asthma is
characterized by variable airflow obstruction, AHR, and airway
inflammation. Although asthma is multifactorial in origin, it has been
suggested that T lymphocytes, and in particular
CD4+ T cells producing a Th2 pattern of
cytokines, have a prominent effect in the pathogenesis of this disease
(16, 21). To examine the possible anti-inflammatory
effect of low dose IFN- on allergic airway reactivity, we used a
treatment protocol of i.p. injection of 6 U/day (0.25 U/g) of
IFN-, beginning on the first day of OVA inhalation. As can be seen
in Fig. 2A, the inhalation of
aerosolized OVA caused the infiltration of eosinophils
(48.9% OVA/OVA vs 0.3% control; p = 0.0026) and
lymphocytes (14.5% OVA/OVA vs 3.4% control) into the BAL of
OVA-sensitized mice, while a low dose of IFN- dramatically inhibited
the Ag-induced eosinophil (16.8% IFN- vs 48.9%
OVA/OVA; p = 0.01) and lymphocyte (8.7% IFN- vs
14.5% OVA/OVA) recruitment into the BAL. Moreover, low levels of
IFN- dramatically inhibited homing of lymphocytes to the lung LNs
(Fig. 2B).
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Penn for the early
reaction 0.93; baseline 0.092 vs OVA/OVA early 0.185; p
= 0.0002). Furthermore, the airway responsiveness of the
OVA/OVA-challenged mice was significantly augmented to the late Ag
challenge ( Penn 0.52). However, no AHR was observed in the early
reaction ( Penn 0.04) or late reaction ( Penn 0.02) of control
(nonchallenged) mice. Treatment of the OVA/OVA-challenged mice with low
dose IFN- significantly reduced AHR (OVA/OVA 0.185 vs IFN- 0.113
early; p = 0.000028). Penn for early reaction was
significantly reduced by 79% ( Penn early reaction OVA 0.93 vs
Penn early reaction IFN- 0.2). Moreover, IFN- reduced the late
AHR in OVA-challenged mice (OVA/OVA 0.14 vs IFN- 0.11;
p = 0.03). Penn for late reaction was significantly
reduced by 59% ( Penn late reaction OVA 0.48 vs Penn late
reaction IFN- 0.2) (Fig. 2C).
Histopathologic examination of lung tissue from OVA mice revealed a
pleomorphic peribronchial and perivascular infiltrate consisting of
eosinophils, lymphocytes, macrophages, and neutrophils that
was not seen in control mice (Fig. 3).
The peribronchial and perivascular inflammatory infiltrates were given
an inflammatory score of between 1 and 4 by two different viewers. The
inflammatory infiltrate in the OVA/OVA-treated mice was significantly
increased as compared with the control animals (OVA/OVA 3 ± 0.86
vs control 0.11 ± 0.33; p = 7 x
10-8). Lung histological changes in low dose
IFN--treated mice were barely detectable, with evidence of only
slight accumulation of inflammatory cells around the bronchioles. The
peribronchial inflammatory infiltrate was significantly reduced in
these IFN--treated mice (IFN- 0.88 ± 0.54 vs OVA/OVA 3
± 0.86; p = 0.000013).
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dramatically reduced this T cell
infiltration (Fig. 3). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was initially identified based on its antiviral
activities, and is now known to play a much more important role as a
proinflammatory molecule (22). Previously, we showed that
low levels of IFN- down-regulate homing of B cells to the LNs but
not to the spleen (11). The data presented in this study
demonstrate that low levels of IFN- inhibit similar processes of T
cells and as a consequence, down-regulate the Th2 inflammatory
response.
Low dose IFN- inhibits adhesion and transwell migration of naive T
cells and of Th2 cells in vitro. The effect of IFN- on naive T cells
is concentration-dependent with a maximum inhibition of migration and
cytoskeleton rearrangement at 0.1 U/ml. Moreover, our results
demonstrate an inhibitory in vivo effect of low levels of IFN-. Low
levels of this cytokine inhibit the migration of labeled naive cells to
the LNs and decreases the AHR, airway inflammation, and
eosinophil recruitment to the bronchial airways in the
asthma Th2 model. Thus, IFN-, in addition to acting as a local
proinflammatory cytokine, may under specific conditions exert
counterinflammatory effects on T cells. It is possible that exposure of
circulating effector cells in the blood or the spleen to low levels of
IFN- serves to suppress their migratory capacities. We suggest that
the profound inhibition observed in the in vivo model might result from
the combined effect of low levels of IFN- on various T cell
populations. These low levels of IFN- down-regulate homing of naive
T cells to the LNs and thereby reduce the exposure of these T cells to
Ag, and as a consequence their activation is prevented. In addition,
these low levels of IFN- dramatically inhibit homing of effector Th2
cells to sites of inflammation where they exert their function. Thus,
IFN- has a dual function in the asthma model, both at sensitization
and effector stages, which results in a dramatic inhibition on the
inflammatory response.
Several studies have previously analyzed the effect of IFN- on
asthma. Targeted disruption of the IFN- receptor gene results in a
prolonged airway eosinophilia in response to allergen
(23). In addition, i.p. administration of rIFN-
decreases the OVA-induced eosinophil infiltration in a
dose-dependent manner with a significant minimum effect at 600
U/day and almost complete inhibition at doses higher than 20,000
U/day (24). These relatively high doses of IFN-, which
were considered to function in the transition from Th2 to Th1 cells,
can potentiate the effects of other proinflammatory mediators. Our
results show that low levels of IFN-, if properly targeted, might
have a clinical application as an anti-inflammatory agent.
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Acknowledgments |
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Footnotes |
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2 L.F. and I.T. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Idit Shachar, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: Idit.Shachar{at}weizmann.ac.il
4 Abbreviations used in this paper: LN, lymph node; ECM, extracellular matrix; AHR, airway hyperactivity; BAL, bronchoalveolar lavage; Penn, enhanced expiratory pause; SDF-1, stromal-cell-derived factor 1.
Received for publication January 15, 2002. Accepted for publication February 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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negatively regulates homing of immature B cells. J. Exp. Med. 192:1381. down-regulate the integrin-dependent adhesion of B cells by inhibition of cytoskeleton rearrangement. J. Biol. Chem. 276:46701. regulate allergic airway inflammation and mucus production. J. Exp. Med. 190:1309. signaling alters the function of T helper type 1 cells. J. Exp. Med. 192:977. and its receptor. Annu. Rev. Immunol. 11:571.[Medline]
receptor have impaired ability to resolve a lung eosinophilic inflammatory response associated with a prolonged capacity of T cells to exhibit a Th2 cytokine profile. J. Immunol. 156:2680.[Abstract]
regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells. J. Exp. Med. 177:573.This article has been cited by other articles:
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