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*
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington, New Zealand;
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543; and
Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Sydney, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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11+V
3+ transgenic TCR
specific for I-Ek and moth cytochrome c.
Treatment with Y100F-Ig inhibited the induction of lung eosinophilia in
adoptively transferred mice. However, Y100F-Ig did not detectably
affect the accumulation of Ag-specific T cells at the site of peptide
deposit or in the draining lymphoid tissues. Acquisition of an
activated phenotype and expression of adhesion molecules required for
migration into the lung were modestly affected. Importantly, treatment
with Y100F-Ig diminished the ability of T cells to produce the
cytokines IL-4 and IL-5 following intranasal challenge with Ag. All the
responses examined were severely inhibited by treatment with CTLA4-Ig.
We conclude that T cells require CD80 costimulation for the optimal
production of IL-5 following intranasal administration of Ag. Decreased
IL-5 production is the most likely explanation for the diminished
airway eosinophilia observed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously investigated the role of CD80 costimulation during in vivo T cell responses using a mutant form of CTLA4-Ig, Y100F-Ig. Y100F-Ig and CTLA4-Ig bind equally well to CD80, but the binding of Y100F-Ig to CD86 is undetectable (6). Consequently, Y100F-Ig can be used to selectively block CD80 costimulation. In a model of chicken OVA-induced airway eosinophilia, we have shown that treatment with Y100F-Ig suppressed eosinophil and, to a lesser extent, lymphocyte infiltration into the lung of OVA-challenged mice, but had no effect on systemic blood eosinophilia or Ab production (6, 7).
There are several possible explanations for the ability of Y100F-Ig to suppress OVA-induced lung inflammation. Y100F-Ig treatment may limit the activation and/or clonal expansion of Ag-specific T cells in secondary lymphoid tissues, such that responses in the lung can no longer be induced. Alternatively, CD80 blockade may result in altered regulation of the adhesion marker expression or the chemokine production necessary for the migration of CD4+ Th2 cells into the lungs of airway-challenged mice. Lastly, CD4+ Th2 cells may be able to migrate into the lungs of airway-challenged mice, but CD80 blockade may prevent T cell activation at this site, resulting in decreased local IL-5 and cytokine production.
We have developed a new model of Ag-induced lung eosinophilia mediated by peptide-specific T cells expressing a transgenic TCR. Using this model we can identify the T cells responsible for mediating airway inflammation by specific mAbs and investigate the effect of Y100F-Ig treatment on peptide-induced T cell activation and migration in vivo. This has enabled us to clarify the mechanism by which Y100F-Ig suppresses lung inflammatory responses while leaving systemic eosinophilia and Ag-specific Ab production intact.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were bred and maintained at the animal facility of the Wellington School of Medicine. B10.A mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained by brother x sister mating. The -I line 5C.C7 transgenic mice (8, 9) were backcrossed to B10.A mice and maintained by breeding transgenic males to B10.A females. The TCR used to generate the 5C.C7 transgenic strain was derived from the cytochrome c-specific T cell clone 5C.C7. These cells are specific for pigeon cytochrome c fragment 81–104 and I-Ek, but proliferate more vigorously when stimulated with peptide fragment 81–103 from the tobacco horn worm moth cytochrome c (MCC)3 (10, 11). All animal experimental procedures used in this study were approved by the Wellington School of Medicine animal ethics committee and were conducted in accordance with the guidelines of the University of Otago (Dunedin, New Zealand).
Purification of lung lymphocytes, peritoneal cells, and PBLs
Mice used for the preparation of lung lymphocytes were injected
i.p. with 150 U of heparin (Leo Pharmaceutical Products,
Denmark) and sacrificed. The lower vena cava was severed, and
lungs were perfused via the heart right ventricle with 
5 ml of PBS
to remove circulating blood. Minced lung tissue was incubated for 30
min in complete IMDM containing 2.4 µg/ml collagenase type II (Life
Technologies, Auckland, New Zealand) and 0.1% DNase I (Sigma,
St. Louis, MO). Complete IMDM consisted of IMDM (Sigma) with 5% FCS
(Life Technologies), 2 mM glutamine (Sigma), 1%
penicillin-streptomycin (Sigma), and 5 x
10-5 M 2-ME (Sigma). Remaining tissue was then
broken down by passage through an 18-gauge needle, and mononuclear
cells were purified by gradient centrifugation over Lympholyte-M
(Cedarlane Laboratories, Ontario, Canada) according to the
manufacturer’s instructions. Macrophages were depleted by culturing
cells on plastic dishes in complete IMDM for 1–2 h at 37°C.
Peritoneal lymphocytes were obtained by sacrificing mice and flushing
10 ml of PBS into the peritoneal cavity three times. PBLs were
purified by collecting 100 µl of tail blood into 1 ml of Alsevers’
solution (2% dextrose, 0.4% NaCl, and 0.8% sodium citrate) and
lysing RBC by incubation in 0.14 M NH4Cl and 17
mM Tris-HCl for 10–20 min at 37°C.
FACS staining
FACS analysis of lymphocytes was conducted by staining in
96-well round-bottom plates at
105–106 cells/well for
10–15 min on ice using the appropriate mAbs diluted in 100 µl of
FACS buffer (PBS plus 2% FCS and 0.01% sodium azide). 2.4G2 (10
µg/ml) was used to inhibit Fc
RII-mediated uptake. Flow cytometric
analysis was performed on a FACSort (Becton Dickinson, Mountain View,
CA) using CellQuest software.
Reagents and mAbs
CTLA4-Ig, Y100F-Ig, and L6-Ig were purified from culture medium
of stably transfected Chinese hamster ovary cells as previously
described (6). Anti-CD4 (GK1.5) was grown from hybridoma
culture supernatant and conjugated to FITC or biotin. Anti-V11-FITC,
anti-V3-PE, anti-V3-biotin, anti-CD49d-PE,
anti-CD44-PE, anti-CD62L-PE, anti-CD80-FITC, anti-CD86-PE,
and streptavidin-FITC, -PE, and -Cy-Chrome were all obtained from
PharMingen (San Diego, CA).
Ag-induced airway inflammation
The protocol for induction of OVA-dependent airway inflammation
has been described previously (6). For the adoptive
transfer model, cell suspensions were prepared from lymph nodes of
5C.C7 mice, and the percentage of V11, V3 TCR-expressing
lymphocytes was determined by FACS analysis. A total of 3 x
106
V11+V3+ T cells were
injected into the tail vein of sex-matched B10.A mice in a total volume
of 500 µl of IMDM. Two days later recipient mice were immunized i.p.
with 250 µg of MCC87–103 in 200 µl of alum
adjuvant (SERVA, Heidelberg, Germany) on days 0 and 20 after adoptive
transfer. Six days after the last i.p. immunization, mice were
anesthetized by injection of a mixture of ketamine and xylazine
(Phoenix, Auckland, New Zealand) and challenged with 100 µg of
MCC87–103 in 50 µl of PBS by intranasal (i.n.)
inoculation. At the corresponding times, nonimmunized mice received
alum adjuvant i.p. and PBS i.n. Mice were sacrificed at the indicated
time points after i.n. challenge, and cellular infiltration into the
airways was determined by bronchoalveolar lavage (BAL) and differential
cell counting as previously described (6).
Enzyme-linked immunospot (ELISPOT)
Cells were cultured at various dilutions for 6 h at 37°C
in 96-well plates (Nunc, Copenhagen, Denmark) coated with 10 µg/ml
anti-CD3 mAb (2C11) and 20 µg/ml anti-IL-5 mAb (TRFK5),
anti-IL-4 mAb (11B11), or anti-IFN- mAb (R4-6A2) in the
presence of IL-2 (10 U/ml). As a control, cells were also incubated on
plates coated with the capture Ab only. After the initial 6-h
incubation plates were washed thoroughly with PBS-Tween, then 5 µg/ml
biotinylated detection Abs (anti-IL-5 mAb, TRFK4; anti-IL-4
mAb, BVD6-24G2; anti-IFN- mAb, xMG-D6) were added, and plates
were incubated overnight at 4°C. Washed plates were then incubated
with avidin-alkaline phosphatase conjugate (Sigma) for 1 h at room
temperature. Reactions were developed using
5-bromo-4-chloro-3-indoyl-phosphate (Sigma) and were stopped by
addition of distilled water. The number of spots was determined by
counting under an inverted microscope.
RNA isolation and cDNA preparation.
RNA was isolated from mediastinal lymph node (MLN) and lung using TRIzol (Life Technologies). The RNA was quantified using GeneQuant, and 1 µg of total RNA was used in the cDNA synthesis reaction. The cDNA reaction was conducted for 60 min at 37°C using 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies) and 0.5 µg of oligo-(dT)12–18 primer (Life Technologies).
Polymerase chain reaction
Primers and probes for IL-4, IL-5, IFN-, and
2-microglobulin were designed and synthesized
as described previously (12). Probes were modified to
incorporate a reporter dye at the 5' end (6-carboxy-fluorescein or
tetracholoro-6-carboxy-fluorescein) and a quencher at the 3' end
(6-carboxy-tetramethyl-rhodimine). The sequences of the
oligonucleotides and PCR conditions are detailed in the report by Hook
et al. (12). PCRs were conducted in an ABI Prism 7700
Sequence Detector (PE Applied Biosystems, Foster City, CA). Reactions
were set up using the TaqMan core reagents (PE Applied Biosystems)
according to the manufacturer’s instructions.
MgCl2 (5 mmol/L) was used in the IL-4, IL-5,
IFN-, and 2-microglobulin reactions.
Cycling conditions consisted of 50°C for 2 min, 94°C for 10 min,
and then 35 cycles of 94°C for 15 s, 58°C for 30 s, and
72°C for 30 s. DNA standards were made for each of the cytokines
and serially diluted and included with each PCR. Data were analyzed
using ABI Prism Sequence Detection System version 1.63 software (PE
Applied Biosystems). All amplifications were performed in duplicate,
and the amount of DNA in the samples was calculated from the standard
curve. All results were normalized to
2-microglobulin to compensate for differences
in the amount of cDNA in the samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A model of peptide-induced lung eosinophilia was developed using
the adoptive transfer of T cells from 5C.C7 mice (8, 9),
which are transgenic for a
V11+V3+ TCR specific
for I-Ek and MCC87–103
(10, 11).
Initial experiments were conducted to determine the optimal
immunization conditions for the induction of lung eosinophilia. The
variables examined included the number of adoptively transferred T
cells, the peptide dose, and the number and timing of i.p.
immunizations. It was found that a large number of adoptively
transferred TCR transgenic T cells induced a good response, but was
associated with occasional lethality at the time of i.n. challenge.
Moreover, i.p. immunizations with larger amounts of peptide and at
longer intervals appeared to yield more substantial and more
reproducible eosinophil infiltration (data not shown). Fig. 1
outlines the optimal protocol as
determined in those experiments. Briefly, 3 x
106 5C.C7 T cells expressing the transgenic
V11+V3+ TCR were
adoptively transferred into normal syngeneic B10.A mice 2 days before
the first immunization. Recipient mice were immunized i.p. with 250
µg of MCC87–103 in alum adjuvant on days 0 and
20, and then given an i.n. challenge with 100 µg of
MCC87–103 in PBS on day 26. Cellular
infiltration into the airways and lung was determined by BAL.
Importantly, no airway eosinophilia was observed in B10.A mice that
were immunized and airway challenged with
MCC87–103, but had not received T cells
expressing the transgenic
V11+V3+ TCR, using
these immunizing conditions.
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To determine the effect of Y100F-Ig treatment on
MCC87–103-induced airway eosinophilia B10.A mice
were adoptively transferred with
V11+V3+ T cells from
5C.C7 donors, then immunized with MCC87–103/alum
and airway challenged as detailed in Fig. 1
. Mice were treated with
i.p. injections of 400 µg of human or 200 µg of murine CTLA4-Ig,
Y100F-Ig, or the control molecule L6-Ig every 48 h, beginning on
the day of the first MCC87–103 i.p.
immunization.
The BAL fluid obtained from mice treated with the isotype control L6-Ig
contained high numbers of eosinophils and lymphocytes. Treatment with
CTLA4-Ig completely abrogated eosinophil and lymphocyte infiltration
into the BAL fluid of MCC87–103-challenged mice
(Fig. 2). Treatment with Y100F-Ig
decreased the number of eosinophils in the BAL 3- to 5-fold on days 1,
3, and 5 after i.n. challenge (Fig. 2), but did not alter
MCC87–103-induced lymphocyte infiltration into
the BAL (Fig. 2). Although there was some variability in the
MCC87–103-induced eosinophilia, the suppressive
effect of Y100F-Ig treatment was statistically significant over all
time points using ANOVA of log data (p =
0.0001).
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Treatment with Y100F-Ig does not alter the accumulation of
V11+V3+ T cells in the secondary lymphoid
tissues or at the site of Ag deposit
A possible explanation for the observation that Y100F-Ig can
suppress airway eosinophilia is that CD80 costimulation is required for
optimal activation and/or clonal expansion of Ag-specific T
cells following i.p. immunization. Alternatively, CD80 blockade may
suppress lung eosinophilia by altering T cell responses following the
i.n. challenge of sensitized mice. To distinguish between these
possibilities, the activation and accumulation of T cells expressing
V11+V3+ TCR were
determined at different times after the second i.p. immunization and
after the i.n. challenge.
After the second i.p. immunization with
MCC87–103 in alum, a considerable accumulation
of V11+V3+ cells was
observed in the spleen and peritoneal cavity of immunized mice compared
with nonimmunized controls (Fig. 3A). A detectable, but
smaller, accumulation of
V11+V3+ T cells was
also observed in the lymph nodes of immunized mice (data not shown).
Treatment with Y100F-Ig had no effect on the number of
V11+V3+ T cells that
accumulated in the spleen or peritoneal cavity (Fig. 3A). In
contrast, treatment with CTLA4-Ig abrogated
MCC87–103-induced accumulation of
V11+V3+ cells at both
sites (Fig. 3A). Therefore, these data do not support the
possibility that Y100F-Ig treatment alters the clonal expansion or
site-specific accumulation of Ag-reactive T cells following i.p.
immunization.
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11+V3+ T cells in
the MLN, lung, and BAL fluid following i.n. challenge with
MCC87–103 peptide. As shown in Fig. 3B, treatment with Y100F-Ig did not notably alter the
MCC87–103-induced accumulation of
V11+V3+ T cells at
any of those sites. In contrast, treatment with CTLA4-Ig inhibited the
accumulation of
V11+V3+ T cells at
all sites investigated (Fig. 3B). Thus, these data do not
support the alternative possibility that Y100F-Ig treatment may prevent
the specific accumulation of Ag-reactive T cells following i.n.
administration of Ag.
The expression of activation markers was examined on Ag-specific T
cells in the MLN after i.n. challenge with
MCC87–103. In immunized mice,
V11+V3+ T cells
expressed significantly decreased levels of CD62L (L-selectin) compared
with the same cells in naive 5C.C7 mice, while their expression of CD44
(PgP-1) was increased (Fig. 4). This
phenotype is characteristic of Ag-activated T cells. Treatment with
CTLA4-Ig inhibited almost completely the progression of
V11+V3+ T cells from
the naive to the activated phenotype (Fig. 4). In contrast,
V11+V3+ T cells in
mice treated with Y100F-Ig clearly showed an activated phenotype in
terms of CD62L and CD44 expression. However, on close examination the
ratio of activated, CD62Llow
CD44high, to naive
CD62Lhigh CD44low
V11+V3+ T cells was
slightly lower in the Y100F-Ig group compared with that in the control,
L6-Ig-treated group.
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11+V3+ T cells
following MCC87–103 immunization and airway
challenge, but does result in a slightly less activated phenotype
compared with that of control, L6-Ig-treated mice. The likely outcome
of a small change in activation marker expression in terms of T cell
effector function is unknown.
Treatment with Y100F-Ig does not prevent the depletion of
V11+V3+ T cells from peripheral blood
following i.n. challenge with MCC87–103 or expression of
integrins involved in migration into inflammatory sites
The data presented in the previous section demonstrate that
treatment with Y100F-Ig did not affect the accumulation of
V11+V3+ T cells in lymphoid organs and
lung after i.p. immunization and i.n. challenge with
MCC87–103. The expression of the activation markers CD62L
and CD44 was moderately affected.
T cell migration and recruitment can also be examined by determining
number of V11+V3+ T
cells in peripheral blood at various times throughout the
MCC87–103 immunization and airway challenge
protocol. Over a large number of experiments we have observed that i.p.
immunization with MCC87–103 in alum results in
an increase in the percentage of
V11+V3+ T cells in
peripheral blood.
V11+V3+ T cells
rapidly disappear from the blood following i.n. challenge with
MCC87–103 (Fig. 5A), presumably reflecting
migration of activated T cells into the lung and airways. As shown in
Fig. 5A, treatment with Y100F-Ig did not affect the
accumulation of
V11+V3+ T cells in
peripheral blood following i.p. immunization. Y100F-Ig treatment also
did not affect the depletion of
V11+V3+ T cells from
peripheral blood following i.n. challenge with
MCC87–103 (Fig. 5A). In contrast,
accumulation of
V11+V3+ T cells in
the blood was completely inhibited by treatment with CTLA4-Ig,
preventing further analysis of their migration.
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subunit, CD49d, on
V11+V3+ T cells from
i.p. immunized mice. No CD49d-expressing
V11+V3+ T cells could
be demonstrated in the lymph nodes or spleen of immunized mice (not
shown). In control L6-Ig-treated mice peripheral blood
V11+V3+ T cells
expressed increased levels of CD49d compared with
V11+V3+ T cells from
naive 5C.C7 transgenic mice (Fig. 5B). Cells expressing high
levels of CD49d were preferentially depleted from blood after i.n.
challenge with MCC87–103 (data not shown).
Treatment with Y100F-Ig did not alter the
MCC87–103-induced increase in CD49d expression
on blood V11+V3+ T
cells (Fig. 5B). In contrast,
V11+V3+ T cells in
CTLA4-Ig treated mice expressed only low levels of CD49d (Fig. 5B), comparable to the levels observed in naive 5C.C7
mice.
Taken together, the data in Fig. 5 indicate that Y100F-Ig treatment
does not prevent the emigration of
V11+V3+ T cells from
the blood upon i.n. Ag challenge, nor does it alter their expression of
the integrins required for entry into the lung and airways. These data
support the conclusion in the previous section that Y100F-Ig treatment
does not prevent the homing of
V11+V3+ T cells to
the lungs after i.n. administration of Ag.
Treatment with Y100F-Ig inhibits T cell cytokine production
Local IL-5 production is critical to the development of lung
eosinophilia (7, 14). Thus, Y100F-Ig may lower airway
eosinophilia by preventing T cell activation and local IL-5 production
following re-encounter with specific Ag in the lung and airways. We
investigated the effects of L6-Ig, Y100F-Ig, and CTLA4-Ig treatment on
the ability of T cells to produce cytokines following Ag immunization
and airway challenge. However, despite extensive analysis at different
time points after i.n. challenge with MCC87–103
peptide, little cytokine production by
V11+V3+ T cells could
be demonstrated in the lung or lymph node by analysis of ELISPOT or
bulk culture supernatants. We next attempted to determine the effects
of L6-Ig, Y100F-Ig, and CTLA4-Ig treatment on T cell cytokine
production in mice immunized and i.n. challenged with the whole protein
Ag OVA. These experiments were conducted using B10.A mice that had been
immunized and i.n. challenged with OVA as previously described
(6), as this immunization protocol induces a T cell
response in which ex vivo cytokine production can be more easily
measured. As seen using the MCC87–103-induced
airway eosinophilia model, Y100F-Ig treatment of OVA-immunized B10.A
mice resulted in a reduction of airway eosinophilia. In contrast, BAL
lymphocyte numbers were similar in the L6-Ig- and Y100F-Ig-treated
groups. CTLA4-Ig treatment inhibited infiltration of either eosinophils
or lymphocytes into the airways (data not shown).
The numbers of cytokine-producing cells were evaluated using an ELISPOT
assay. Given the short restimulation period required, we considered
that the cytokine production measured by this technique is likely to
reflect cytokine production in vivo. Significant numbers of lymphocytes
producing IL-4 and IL-5 were found in the lung, airways, and MLN of
OVA-immunized and airway-challenged B10.A mice on day 4 following i.n.
challenge (Fig. 6, L6-Ig-treated group).
The numbers of IL-4- and IL-5-producing cells were reduced in the
airways, lung, and MLN of Y100F-Ig-treated mice and were virtually
absent from these tissues in CTLA4-Ig-treated mice (Fig. 6). The number
of IFN--producing cells in the lung and MLN of OVA-immunized and
challenged mice did not differ dramatically from that in nonimmunized
mice (Fig. 6). The numbers of IFN--secreting cells found in the BAL
fluid were increased in immunized, L6-Ig-treated mice, and this
increase was reduced by treatment with either Y100F-Ig or CTLA4-Ig
(Fig. 6). However, it is not clear whether the IFN- produced was
derived from CD4+ or CD8+ T
cells.
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). In parallel with the ELISPOT data,
treatment with either Y100F-Ig or CTLA4-Ig led to a reduction in the
production of IL-4 mRNA in these tissues (Fig. 7). No IL-5 or IFN-
could be detected using this assay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We do not think that these data are in conflict with our previous findings that Y100F-Ig treatment lowered airway eosinophilia, but not blood eosinophilia or Ab production, in immunized mice (6). Rather, these data may simply reflect differences in the level of T cell cytokine production required for the development of airway eosinophilia as opposed to the development of peripheral blood eosinophilia or the provision of T cell help to B cells. For instance, IL-5 production is reduced, but not lost, in Y100F-Ig treated mice. Therefore, the amount of Ag-specific IL-5 production occurring during the immunization and challenge of Y100F-Ig-treated mice may be adequate for the differentiation and recruitment of eosinophils from the bone marrow, but not for their recruitment from blood into lung. Likewise, perhaps only a small number of IL-4-producing T cells are required to deliver help to the limited pool of Ag-specific B cells responsible for producing OVA-specific IgE. If this were the case one would not expect to see a comparable reduction in IgE production following a reduction in the numbers of IL-4-producing T cells.
It is interesting that cytokine production by T cells was decreased by
CD80 blockade while other T cell functions, such as clonal expansion
and migration into peripheral tissues, appeared unaffected. This may
reflect a unique dependence of Th2 cell differentiation and/or Th2
cytokine production on CD80 costimulation. IL-4 and IL-5 cytokine
production are considered to be late events in T cell activation
(15), only occurring following a certain number of cell
divisions, and may be more dependent on CD80 costimulation due to the
slow kinetics of CD80 up-regulation on activated APCs
(16). In our system, we detected no evidence of inhibition
of T cell division, as similar numbers of
V11+V3+ transgenic T
cells were found to accumulate in the lungs and lymphoid tissues of
L6-Ig and Y100F-Ig-treated mice. However, more sensitive techniques may
be required to reveal small differences in cell division. It is also
possible that CD80-mediated signals are required for the
differentiation of T cells into Th2 independently of cell division.
This possibility is favored by the observation that expression of
adhesion molecules on T cells was modestly, but detectably, decreased
in Y100F-Ig-treated compared with L6-Ig-treated mice. Lastly, as
further discussed below, Y100F-Ig may simply act by inhibiting the
secretion of cytokines by fully differentiated effector T cells.
Effector T cells in our model were of the Th2 type; thus, the
inhibitory effect was observed on IL-4 and IL-5 secretion. However, we
do not rule out the possibility that a similar inhibition may be
observed on other types of effector T cells and cytokines. Indeed, we
observed that treatment with Y100F-Ig resulted in decreased IFN-
production by BAL lymphocytes in the present study, and in
CD8+ T cells following infection with influenza
virus (17).
An alternative explanation for our findings is that CD80 blockade may decrease cytokine production following administration of soluble Ag i.n., but not following administration of Ag in alum adjuvant i.p. In this case Y100F-Ig treatment would not be expected to affect the development of blood eosinophilia or the production of T cell-dependent Abs following Ag/alum immunization, but it would lower cytokine production following the administration of Ag in PBS as for the i.n. challenge. This possibility has not been directly investigated; however, an obvious explanation for such a scenario would be that APCs up-regulate CD86 following challenge with Ag in an adjuvant such as alum, providing adequate costimulation to the responding T cells even in the absence of CD80. Conversely, encounter with Ag in PBS may not result in comparable CD86 up-regulation, deeming the presence of CD80 necessary for adequate costimulation.
Although the lack of IL-5 production by lung T cells is likely to
result in defective eosinophil recruitment, we have not ruled out the
possibility that this could also occur by altered chemokine production.
Many chemokines regulate eosinophil chemotaxis, the most potent being
RANTES and eotaxin (18, 19, 20). The major cellular sources of
eotaxin are thought to be the epithelium, endothelium, and eosinophils
themselves rather than T cells (19, 20). However, T cells
are known to produce the chemokines RANTES and macrophage inflammatory
protein-1, and this has previously been shown to be CD28 dependent
(21). Although we have not formally investigated the
possibility, it would not be surprising if T cells that are defective
for IL-4 and IL-5 production also fail to produce relevant
chemokines.
In conclusion, we have found that the major effect of blocking CD80 costimulation on T cell function appears to be in the production of cytokines following i.n. challenge with Ag. The lack of IL-5 production by these T cells is likely to be responsible for the inhibition of airway eosinophilia seen in Y100F-Ig-treated mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Franca Ronchese, Malaghan Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand.
3 Abbreviations used in this paper: MCC, moth cytochrome c; i.n., intranasal(ly); MLN, mediastinal lymph node; BAL, bronchoalveolar lavage; ELISPOT, enzyme-linked immunospot; VLA-4, very late Ag 4.
Received for publication May 1, 2000. Accepted for publication January 31, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 inhibits antigen-induced lung eosinophilia but not Th2 cell development or recruitment in the lung. Eur. J. Immunol. 27:155.[Medline]
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), Y100F-Ig (
), or the control
molecule L6-Ig (
) every 48 h, beginning on the day of the first
i.p. immunization. Mice were sacrificed, and differential cell counts
were made on BAL fluid collected at the indicated time points after the
i.n. challenge. Values represent the mean number of cells per
milliliter of BAL fluid ± SE of five individual mice. Values for
the nonimmunized group are given for day 3 only. The data shown are
from one representative experiment. Similar results were obtained in
three separate experiments using four to six mice per group.
, nonstained
samples; 
