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Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in cells of the innate immune system have been well
established, involving microbial pattern recognition by Toll-like
receptors (1, 2). While NK cells, basophils, and mast
cells can produce IL-4 after stimulation, the signals that induce IL-4
production in these cells of the innate immune system are less well
defined (3, 4).
Naive T cells do not produce cytokines such as IL-4 and IFN-, but
learn to express them via an instructed differentiation process
(5, 6). The cytokine microenvironment during the primary
Ag encounter of the naive T cell defines which cytokine the memory
cells generated will express (7). The generation of
IL-4-producing memory T cells (Th2 cells) requires production of IL-4,
either by the cells of the innate immune system or by T cells during
the primary Ag encounter (8). In contrast to cells of the
innate immune system, cytokine production by memory T cells shows
long-term commitment (9, 10). Memory T cells are
programmed to secrete a certain cytokine whenever and wherever they
reencounter Ag. This commitment enables the predictable reproduction of
the cytokine environment that called the immune response into
existence. Moreover, the expandable nature of the Ag-specific memory T
cells permits regulation of the magnitude of the cytokine effector
functions via clonal sizes.
The effective concentration of the cytokine might be fundamentally different when produced by cells of the innate immune system or by T cells. For example, IL-4 regulates Ig class switching in B cells (11, 12). When an Ag-specific Th2 cell interacts with an Ag-specific B cell, the IL-4 will be released in the immediate vicinity of the B cell, reaching very high effective concentrations for this B cell. In contrast to this cognate event, IL-4 released in the absence of direct cell-cell contact is likely to create orders of magnitude lower effective concentrations. The above occurs, first, because the concentration of the cytokine drops with the square of the distance between the secreting cell and the cell to be affected and, second, because IL-4 has a short t1/2 in vivo (13, 14, 15). It still needs to be defined how the different IL-4-dependent mechanisms such as Ab switching to IgE and IgG1, as well as T cell differentiation, are affected when IL-4 is released by memory T cells in cognate cell-cell interactions, vs the indirect pathway production by cells of the innate immune system.
Because Th1 and Th2 cells exert fundamentally different, frequently
antagonistic roles in T cell-mediated immunity, it has been a major
scope of immunologic research to clearly define Th1/Th2 cell
involvement in most aspects of immunobiology and immunopathology.
Measurements of recall Ag-induced IFN- and of IL-4 have been the
gold standard for assessing Th1 and Th2 immunity, respectively. The low
frequency of Ag-specific T cells has made such measurements challenging
in freshly isolated cell material (16), in particular
using techniques that permit to directly verify the phenotype of the
IL-4-producing cell. With few exceptions, in which the specific T cells
reach high enough frequencies for direct flow cytometric detection
(17, 18), judgment on the induction of Th1/Th2 immunity
has relied primarily on measurements of recall Ag-induced IL-4 and of
IFN- in supernatants or lysates of mixed cell populations with the
cellular source of the cytokine not amenable for direct detection.
However, only IL-4 and IFN- produced by T cells (cognate cytokine)
reflect long-term commitment by the immune system, and hence the
sought-after information. In contrast, IL-4 or IFN- production
induced by the recall Ag in cells of the innate immune system reflects
indirect, bystander reactions of unknown consequences as to in vivo
immune processes.
In this study, we show that while recall Ag-induced production of
IFN- is a cognate T cell response in freshly isolated lymphoid cell
material, IL-4 production can reflect a cytokine-driven bystander
reaction of cells of the innate immune system. Therefore, such
measurements on recall Ag-induced IL-4 are unreliable indicators of Th2
T cell immunity. We also have shown that the indirect IL-4 pathway
mediates in vivo immunoregulatory and effector functions distinct from
those resulting from cognate T cell-derived IL-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cByJ, BALB/cByJ Smn-Prkdcscid/J, C57.BL/6J; SJL/J, C57.BL/6J-KitW-v, WBB6F1/J-KitW/KitW-v, IL-4 -/-, and RAG2 -/- (both on C57.BL/6 background) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at Case Western Reserve University (Cleveland, OH) under specific pathogen-free conditions. Ags were mixed with the adjuvants to a final concentration of 1 mg/ml OVA and 1 mM of emulsion (OVA323–339, proteolipid protein (PLP)3139–151, and myelin oligodendrocyte glycoprotein (MOG)35–55). A total of 100 µl of Ag in CFA or alum was injected either i.p. or s.c. Female mice were injected at 6–10 wk of age. OVA was purchased from Sigma-Aldrich (St. Louis, MO). OVA peptide 323–339, PLP peptide 139–151, and MOG peptide 35–55 were purchased from Princeton Biomolecules (Columbus, OH). IFA was purchased from Life Technologies (Grand Island, NY), and CFA was prepared by mixing inactivated Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) at 1 mg/ml into IFA. Alum (Inject Alum) was purchased from Pierce (Rockford, IL). For the induction of experimental autoimmune encephalomyelitis (EAE), mice were injected with PLP139–151 (SJL) or MOG35–55 (C57.BL/6) 100 µg in CFA, and pertussis toxin was injected (0.2 µg) twice, once immediately and once 24 h after immunization (19). The care of mice was in accordance with institutional guidelines.
Measurement of specific serum Abs
Plates (Nunc Immunoplate; Fisher Scientific, Pittsburgh, PA) were coated with OVA (10 µg/ml) overnight at 4°C, then blocked for 1–2 h with 0.1% gelatin, both in PBS containing 0.05% Tween 20 (PBST). The test serum was added and incubated overnight at 4°C. Plate-bound Ab was detected by alkaline-phosphatase-coupled anti-mouse Ig. Affinity-purified goat anti-mouse IgG (H and L chains) from Southern Biotechnology Associates (Birmingham, AL) was used to detect total Ig; the isotype-specific Abs used to detect IgG1 and IgG2a were also from Southern Biotechnology Associates. p-nitrophenyl phosphate disodium was used for the development of the colorimetric reaction.
Cell preparation from organs, cell purifications
Single cell suspensions from draining lymph nodes (dLN) and nondraining LN and the spleen were prepared as previously described (20). CD4 cell fractions were obtained with >97% purity by passing single cell suspensions over mouse CD4 subset columns (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Cells from the CNS were prepared as follows. After sacrificing, the animals were perfused with PBS, and the spinal cords were removed from the entire vertebral column and placed into DMEM medium. The spinal cord was disrupted with the back of a syringe. The resulting cell suspension was filtered through a Falcon Cell Strainer 2350 (BD Biosciences, San Jose, CA). The cells were washed twice with DMEM and subsequently counted. The cells were resuspended in HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1% glutamine and plated typically at 5 x 104–5 x 105 cells/well, or at serial dilutions. Cells from the peritoneal cavity were obtained by lavage after injecting 7 ml of DMEM medium. Mouse blood was obtained by retroorbital bleeding, using heparin as an anticoagulant. The blood was diluted four times with sterile saline, and PBMCs were obtained by density gradient centrifugation over a Ficoll Histopaque 1083 gradient (Sigma- Aldrich). Single cell suspensions from dLN and nondraining LN and the spleen were prepared as previously described (21). For flow cytometric studies of the cell populations obtained, we used the directly labeled Abs specified, all from BD PharMingen (San Diego, CA). The stained cells were analyzed with a FACScan and CellQuest software (BD Biosciences). The cells isolated from spinal cords of mice with EAE contained 19–43% CD4 cells.
ELISPOT assays
Plates (ImmunoSpot M200; Cellular Technology, Cleveland, OH)
were coated overnight at 4°C with the cytokine-specific capture Abs
specified below. The plates were then blocked with 1% BSA in PBS for
1 h at room temperature and washed four times with PBS.
Subsequently, nonirradiated or irradiated (3000 rad) LN or splenic APC
from naive, syngenic mice were added (5 x
105 cells/well, or as specified). Freshly
isolated CD4 cells or single cell suspensions from spinal cords were
plated in serial dilution in two to four replicate wells with or
without the nominal Ag or control Ags. Freshly isolated spleen cells
and LN cells were plated at 106 and 7 x
105 cells/well respectively, or as specified, in
two to four replicate wells with or without the nominal Ag or control
Ags. The assay medium was serum-free HL-1 (BioWhittaker) supplemented
with 1 mM L-glutamine. Following 36 h of cell culture
in the incubator at 37°C, the cells were removed by washing three
times with PBS and then four times with PBST, and biotinylated
detection Abs (as specified below) were added. After overnight
incubation at 4°C, plates were washed three times with PBST, followed
by a 2-h incubation at room temperature with streptavidin-alkaline
phosphatase conjugate (DAKO, Carpenteria, CA) at 1/2000 dilution.
Plates were then washed twice with PBST and twice with PBS. For
two-color assays, two detection Abs were added simultaneously, followed
by incubation with streptavidin-alkaline phosphatase and subsequent
incubation with two substrates. The nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added first.
Then, after washing twice with PBS, the 3-amino-9-ethylcarbozole
substrate (Pierce) was added (15–30 min for nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate and 20–40 min for
3-amino-9-ethylcarbozole). The following coating mAbs were used for
IL-2, IL-3, IL-4, IL-5, and IFN-: JES6-1A12 (5 µg/ml), MP2-8F8 (5
µg/ml), BVD4-1D11 (2 µg/ml), TRFK5 (5 µg/ml), and R46A2 (2.5
µg/ml) (BD PharMingen). For the IL-4:IFN- two-color assay, we used
a combination BVD4-24G2-biotin:XMG1.2-HRP (BD PharMingen). HRP labeling
of Abs was performed by the periodate method (22). The
detection Ab concentrations were as follows: JES6-5H4-biotin (2
µg/ml), MP2-43D11-biotin (2 µg/ml), BVD4-24G2-biotin (2.5 µg/ml),
TRFK4-HRP (2 µg/ml), XMG1.2-HRP (2 µg/ml), and XMG1.2-biotin (2
µg/ml).
Computer-assisted ELISPOT image analysis
The image analysis was performed as previously described (20). Briefly, we used a Series 1 ImmunoSpot Image Analyzer (Cellular Technology) that was customized for two-color analysis. Digitized images were analyzed for the presence of areas in which color density exceeded background by a factor set on the basis of the comparison of control wells (containing T cells and APC without Ag) and experimental wells. After separating spots that touch or partially overlap, additional criteria of spot size and circularity were applied to gate out speckles and noise caused by spontaneous substrate precipitation, nonspecific Ab binding, and ELISA effects. Objects that did not meet these criteria were ignored, and areas that met them were recognized as spots, counted, and highlighted. For two-color ELISPOT image analysis, the analyzer detects red, blue, and double-colored spots separately by using three different threshold settings. Each color threshold is set in red-green-blue mode and consists of three numbers reflecting the threshold in the red, blue, and green channels. The red and blue thresholds are set by using spots from single-color assays. With the blue threshold active, single-color blue and all double-positive spots (which have blue as a part of their color composition) are detected and outlined. Similarly, under the red threshold, only red single-color and double-color spots are detected. The double-color threshold is a mathematical intersection of the two single-color thresholds. In the final step of analysis, single-color and double-color spots are highlighted with artificial colors: blue, red, and green for single-blue, single-red, and double-color spots, respectively. To eliminate the assessment of partially overlapping red and blue spots as double-color spots, the image analyzer counts only double-color spots that are formed by the color mixture of concentric blue and red spots with single dense centers.
Passive cutaneous anaphylaxis assay
This assay was performed as previously described (23, 24). Briefly, sera were obtained from five to seven mice
immunized with OVA in CFA or alum, and pooled. The abdomens of
unimmunized mice were shaved, and 30 µl of immune serum was injected
intradermally in serial dilutions as specified in Fig. 4
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Injections were repeated 24 h later. Three hours after the second
injection, mice were injected i.v. with a mixture of 100 µl of 0.5%
Evans blue dye and 1 mg of OVA. PBS injection was used as a control.
Thirty minutes later, the mice were sacrificed, the skin on the belly
was inverted, and cutaneous reaction was evaluated by the sizes of
visible blue spots. A reaction was scored according to the following
scale: negative, less than 4 mm in diameter; 1, 4 mm; 2, between 4 and
6 mm; 3, between 6 and 10 mm; and 4, >10 mm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been controversial to what extent highly polarized type 1
immunity can be induced by immunization with adjuvants. We revisited
this question, taking advantage of cytokine ELISPOT assays that permit
monitoring cytokine production by individual cells in the frequency
below the detection limit of flow cytometry-based detection systems
(<1:10,000), and at which Ag-specific T cells apparently invariably
occur after immunizations with adjuvants (21). We studied
immunity induced in C57.BL/6 mice after injecting s.c. OVA protein
emulsified in CFA. dLN were isolated 10 days after the immunization and
were challenged directly ex vivo with OVA, using ELISPOT assays to
measure the frequency of cells induced to produce cytokine. Cells
producing IFN-, TNF-
, IL-2, and IL-3 were detected in the 40–100
per million range, while the frequency of cells producing IL-4 or IL-5
was <1 per million (Fig. 1A).
This cytokine hierarchy was invariably seen in the dLN at all time
points measured (days 3–90) at all recall Ag concentrations tested
(using either OVA protein or the I-Ab-restricted
peptide OVA323–339) (data not shown). The cytokine
production was seen in immunized mice only and was recall Ag specific
(21). While the cytokine signature in the dLN was
therefore highly type 1 polarized, measurements done simultaneously on
the spleen gave a different result. In addition to the type 1 cytokines
detected in the dLN, a vigorous IL-4 (but no IL-5) recall response was
detected (Fig. 1B) over a wide range of OVA or
OVA323–339 peptide concentration. This IL-4 response was
not seen in naive C57.BL/6 mice, and was induced by OVA or
OVA323–339 peptide only in OVA-immunized mice (data not
shown and Ref. 20). In all 53 mice studied individually,
differential production of IL-4 was seen between the spleen and the
dLN. Are the T cells in the spleen therefore less differentiated Th0
cells (that coexpress type 1 and type 2 cytokines) than the fully type
1 polarized cells in the dLN?
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are produced by different cells
As we showed previously (20), two-color cytokine
ELISPOT assays permit studying cytokine coexpression by individual
cells. Because in this assay both cytokines are continuously captured
around the secreting cells during the 48-h observation period, the
assay accounts for possible asynchronous production, or cytokine
switching. If the OVA-specific T cells were of a Th0 type, they should
coexpress IL-4 and IFN-, appearing as double-positive cells in a
two-color IL-4/IFN- ELISPOT assay. Performing such assays, we found
that IL-4 and IFN- were produced by different cells (Fig. 1
C). Therefore, because the IL-4 production in the spleen
cell population does not reflect Th0 cell activity, the question
emerged whether the data provide evidence for polarized Th1 and Th2
subpopulations showing different organ distributions/recirculation
patterns (25, 26, 27, 28), with polarized Th2 cells being present
in the spleen but not in the dLN. Alternatively, the recall Ag-induced
IL-4 might not be a T cell product, but might reflect an IL-4 bystander
reaction that the activated Th1 cells induce in a cell type that is
present in the spleen but not in the LN.
IL-4 is induced in non-T, non-B bystander cells by Ag-stimulated memory Th1 cells
To establish unambiguously whether the recall Ag-induced IFN-
and IL-4 detected in the spleen cell population are indeed produced by
T cells, we purified CD4 cells from spleens of immunized wild-type (WT)
mice and tested them on splenic APC obtained from the respective
congenic cytokine knockout mice (Table I). The frequencies of IFN--producing
CD4 cells were in the same range when tested on WT, IFN-, and IL-4
knockout nonirradiated naive splenic APC. These data establish, first,
that all of the recall Ag-induced IFN--producing cells in primary
cell populations are T cells and, second, that the IFN- and
IL-4-/- APC are equally functional in
stimulating Th1 cells.
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-/- splenic APC were
tested. However, IL-4 was not detected when the same cells were assayed
on IL-4-/- APC (Table I). Because the
activation of Th2 cells might have required IL-4 feedback by the APC,
we also tested these CD4 cells on APC derived from LN cells and
thymocytes from WT animals. Unlike with the tests done with WT spleen
cells, the LN and thymic APC of WT mice also did not yield IL-4
production. These data suggest that the IL-4 production by the WT APC
might reflect bystander cell activation. To directly test this
hypothesis, we isolated CD4 cells primed by OVA:CFA immunization in
IL-4-/- mice and tested them on the
aforementioned panel of APC. Also with these CD4 cells, the frequency
of IFN--producing cells was independent of the type of APC used. No
IL-4 spots were seen on IL-4-/- APC (showing
the cytokine specificity of the detection system). Yet when these CD4
cells were tested on WT APC, a relative high frequency of
IL-4-producing cells was seen. These data clearly establish that the
recall Ag-induced IL-4 was not CD4 memory (Th2) cell derived, but
represented a bystander reaction. The notion that the recall Ag-induced
production of IL-4 in CFA-immunized spleen cells is a bystander
reaction was also confirmed by cell separation experiments: when spleen
cells were first activated with OVA for 12 h, followed by the
depletion of CD4 cells before placing the cells into an ELISPOT
assay, the CD4 cell-depleted cell fraction was void of IFN-- and
IL-2-producing cells but still contained the IL-4-producing cells (data
not shown).
The CD4 cells isolated from the OVA-immunized
IL-4-/- mice also induced this bystander
reaction in RAG2-/- splenic APC (Table I). This
result shows that the cell type that produces the IL-4 is not a T, or
B, or NK1.1 cell (that undergoes receptor rearrangement), but a cell
type of the innate immune system that is present in
RAG2-/- mice. The higher magnitude of bystander
IL-4 production by RAG2-/- APC as compared with
WT APC (approximately two to three times) most likely reflects the
higher frequency of the cells of the innate immune system of
RAG2-/- mice vs WT mice.
The frequency of IL-4-producing bystander cells is 
200 per
million
The aforementioned experiments clearly establish that within
a type 1 T cell response, IL-4-producing cells can be activated in a
bystander reaction in the spleen, reaching frequencies that approximate
the frequencies of the Th1 cells themselves that induce this bystander
reaction. However, it remained unclear what the maximal frequency of
the cells capable of the bystander reaction is, and how the magnitude
of the cognate response relates to the bystander IL-4 production. To
address this question, we plated in serial dilution a Th1 clone (SH10,
which is not capable of IL-4 production) (20) along with a
constant number of congenic, unirradiated BALB/c-SCID spleen cells. The
frequency of cells producing IFN- or IL-4 was measured by ELISPOT
after adding Ag. The number of IFN- spots was a linear function of
the number of cloned cells plated; at all cell dilutions 50% of the
SH10 cells plated produced detectable spots (Fig. 2A); also by
intracytoplasmic staining, 50% of the clone produced IFN- (data not
shown). The use of splenic or LN APC yielded the same results. The IL-4
assay provided different results (Fig. 2B). No IL-4 spots
were detected on LN APC. Using splenic APC, the number of IL-4 spots vs
the number of cloned cells plated yielded a hyperbolic curve, reaching
a plateau value at 200 spots/million. Similar curves were seen when
freshly isolated CD4 cells purified from spleen of OVA/CFA-primed
IL-4-/- mice were titrated with constant
numbers of RAG2-/- APC (data not shown). These
data show that the cell type capable of bystander IL-4 production is
absent in the LN, and present in the spleen in the rather low frequency
of 1:5000. While the maximal frequency of the IL-4-producing bystander
cells in the spleen was in the same order of magnitude as the CD4 cells
producing the cognate IFN-, our efforts to detect either cell type
by intracytoplasmic cytokine staining failed, being in a frequency
range not amenable to flow cytometric analysis.
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Supernatants of activated Th1 cells induce bystander IL-4 production
The induction of bystander IL-4 production by activated CD4 cells
could either require direct cell-cell interaction or be triggered by
soluble factors secreted by the T cell. To address this issue, we
obtained 24-h culture supernatants of splenic CD4 T cells from
OVA-immunized IL-4-/- mice. Spleen cells from
RAG2-/- mice, and LN and spleen cells from
naive WT C57.BL/6 mice were cultured with these supernatants for
12 h while measuring IL-4 production in an ELISPOT assay. IL-4
spots were induced in the spleen cells, but not in the LN cells in a
dose-dependent manner (Fig. 3A). Supernatants of naive
spleen cells cultured with OVA did not induce IL-4 production; the
supernatants from immune cells that were cultured in the absence of OVA
also did not trigger IL-4 production. Therefore, secretory products of
Ag-stimulated memory cells caused the bystander IL-4 reaction,
explaining the apparent recall Ag specificity of the IL-4
production.
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We have tested whether recombinant cytokines can induce the
bystander IL-4 reaction. Of the cytokines tested, IL-3 excelled in
inducing IL-4: the maximal number of 200 IL-4 spots/million spleen
cells was induced by 20 ng/ml rIL-3, and 0.5 pg led to 50% maximal
activation (Fig. 3B). This activity was specific, as it
could be blocked by anti-IL-3 Ab (Fig. 3B,
inset). Only at high concentrations (>500 ng/ml) did IL-2,
TNF-, and IFN- induce IL-4 production, and the frequencies
induced were 10% of that triggered by IL-3 (Fig. 3C).
The type 2 cytokines IL-10 and IL-5 did not induce IL-4 production.
Therefore, the recall Ag-induced production of IL-3 and of other type 1
cytokines by Th1 cells (Fig. 1) can account for the IL-4 bystander
reaction seen in the spleen. IL-3 is known to be a major activation
factor of basophils/mast cells. To test whether this cell lineage is
the one induced to produce IL-4, we also tested spleen cells from two
different strains of mast cell-deficient mice (Fig. 3D). The
frequencies of IL-4-producing cells in the spleen were about 3-fold
lower in these mice after stimulation with rIL-3, as compared with the
respective congenic WT control mice. The data show that mature
basophils/mast cells constitute the majority of the cells engaged in
the IL-4 indirect pathway, but might not be the only cell type capable
of this reaction.
Increased OVA:CFA-induced type 1 immunity in IL-4-/- mice
The frequencies of OVA-specific IFN--producing T cells, and the
titers of OVA-specific IFN--dependent IgG2a/c Abs permit assessing
the magnitude of the specific type 1 immunity induced in vivo, after
immunization with CFA. Because there is essentially no cognate IL-4
production by OVA-specific T cells after immunization with OVA:CFA,
differences in magnitudes of type 1 response in
IL-4-/- and WT mice can be attributed to the
bystander pathway. The frequency of OVA-specific T cells was 2- to
3-fold higher in IL-4-/- mice than in WT
C57.BL/6 mice (Fig. 4A).
Similarly, the levels of IgG2a/c Abs were higher in the
IL-4-/- animals (Fig. 4B). These
data provide evidence that in vivo, the IL-4 bystander reaction can
play a role regulating type 1 immunity.
Bystander IL-4 causes Ab switching to IgG1, but not IgE
Isotype switching to IgG1 and to IgE is IL-4 dependent (11, 12). Because after CFA immunization there is no detectable
cognate IL-4 production by CD4 cells (Fig. 1), measuring these Ab types
should yield information about the functional consequences of bystander
IL-4 production on humoral immunity. After immunization with OVA:CFA,
IL-4-/- mice produced low levels of specific
IgG1 Abs, as compared with WT mice (Fig. 4C). In the latter,
the specific IgG1 levels were similar or moderately higher than after
immunization with alum, which induced CD4 cells that produce IL-4
(21). In the absence of cognate IL-4 production by T
cells, the bystander IL-4 reaction therefore sufficed to bring about
isotype switching to IgG1. In contrast, specific IgE Abs were detected
only in OVA:alum-immunized mice, in whom IL-4-producing T cells were
induced (Fig. 4D); the bystander IL-4 reaction was
insufficient to bring about isotype switching to IgE production. The
two IL-4-dependent processes were therefore differentially sensitive to
the bystander IL-4 reaction, which added features of a partial type 2
Ab phenotype to a T cell response that was essentially pure type
1.
In EAE, the bystander IL-4 production is induced in the spleen and in the blood, but not in the thymus and CNS
To extend the study of indirect IL-4 production to the immune
periphery, we studied PLP139–151-induced EAE in SJL mice. Cells
separated directly from the CNS of animals with clinical EAE can
provide insight into whether the indirect pathway of Ag-specific IL-4
production may play a role in the regulation of the immunopathology of
EAE and other autoimmune diseases. Confirming our previous findings,
high-frequency peptide-specific, IFN--producing T cells could be
detected in dLN, spleen, blood, CNS, and the peritoneal lavage
(29). Fig. 5 shows that also
peptide-induced IL-3-producing cells occurred in relatively high
numbers in these compartments. However, the IL-4 bystander reaction was
primarily seen in the spleen and in the blood only, but not in the CNS
and the dLN. Similar data were obtained in C57.BL/6 mice immunized with
MOG35–55 peptide (data not shown). These data suggest that the
anatomic compartmentalization of the cell type(s) mediating the
indirect IL-4 reaction affects different site-specific processes
differently.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4) and terminally differentiated Th1
and Th2 cells (that express either IFN- or IL-4)
(30, 31, 32, 33, 34). When using the cytokine signature as of recall
Ag-stimulated cell populations directly ex vivo, the immunity induced
by the classic OVA:CFA immunization displayed a pure Th1 phenotype in
the dLN, but a mixed Th0 cytokine signature in the spleen, apparently
prompting different conclusions as to the T cell immunity induced.
Similarly, the studies of the Abs that are typically used to assess
Th1/Th2 immunity provided apparently inconclusive results.
IFN--dependent IgG2a/c Abs (12, 35, 36, 37, 38, 39) were induced,
confirming the type 1 component, but as far as the IL-4-dependent Ab
classes (11, 12, 40) are concerned, IgG1 was present in
high titers, while IgE was not induced.
We studied the cytokine signatures of freshly isolated individual cells
using ELISPOT assays. In this approach, T cells are challenged with Ag
for 24 h, a time period sufficient to induce cytokine expression
in differentiated T cells, but insufficient to permit proliferation and
cytokine differentiation in vitro (5). The assays that we
used, including those for IL-4 and IFN-, have established single
cell resolution (20). Unlike IL-4 measurements in culture
supernatants by ELISA, which are prone to underestimate IL-4 production
due to receptor-mediated capture (15, 41, 42), ELISPOT
assays can reliably detect even one in 1 million IL-4-producing cells
(20). Using this high resolution, the OVA:CFA-induced
response qualified as a highly polarized Th1 response. In addition to
IFN--, TNF--, IL-2-, and IL-3-producing CD4 cells that were
detected in the 50–100/million frequency range, the numbers of
IL-4- or IL-5-producing CD4 cells were <1/million. Therefore, when
correcting for the bystander IL-4 response in the spleen, the T cell
response was at least 50-fold biased toward numbers of T cells
producing type 1 cytokines. Strikingly, type 1 cytokine-producing CD4
cells, in the absence of cognate IL-4-producing CD4 cells, were seen as
early as 3 days after the immunization, at all Ag doses, and
irrespective of the type of APC used in the assays (data not shown and
Ref. 20). Therefore, this type 1 cytokine signature seems
to be acquired without the T cell passing through an extended Th0
stage. Therefore, these data show that, as far as T cell responses are
concerned, polarized type 1 immunity can be readily induced by the
choice of adjuvant alone.
In the absence of cognate IL-4 production by CD4 cells, specific IgE was not produced, but IgG1 was. The notion about the indirect pathway of the induction of IL-4 response having an effector function on IgG1 isotope switching without affecting the IgE switch should help in interpreting partial type 2 Ab phenotype even in face of a T cell response that is essentially pure type 1. In addition to the growing complexity regarding the cellular nature of type 1 immunity itself (whether individual type 1 cytokines are expressed by the individual T cells as cassettes (32, 33, 34), stochastically (43, 44), or in a mutually exclusive manner (5, 20, 45)), this indirect IL-4 pathway adds an additional layer of complexity to type 1 immunity in vivo.
The observed differences in the effect of bystander vs cognate IL-4 on
B cell differentiation most likely reflect effective cytokine
concentrations depending on the way the cytokine was delivered (Fig. 6). For cytokines that have short
t1/2 in vivo, such as IL-4
(14), the targeted release of the cytokine during the
direct interaction of the T cell with the target cell (e.g., the
Ag-specific B cell) will reach local concentrations that are orders of
magnitudes higher than present in the intercellular space after the
release of IL-4 by bystander cells, due to the dilution, degradation,
and capture of the IL-4 by receptors (13). Moreover,
cognate T cell Ag recognition on B cells involves the transmission of
signals through costimulatory molecules, which in conjunction with the
cytokine concentration may have fundamentally different effects on B
cell differentiation and isotype switching (46).
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However, our studies provided evidence for an immune modulatory role of
the IL-4 bystander pathway in the inductive phase of the Th1 response.
After immunization with OVA:CFA, IL-4-/- mice
developed a higher frequency of OVA-specific IFN--producing T cell
population, as compared with the WT C57.BL/6 mice (Fig. 4A),
and they also generated higher serum levels of specific IgG2a/c Abs
(Fig. 4B). Because there are essentially no cognate
IL-4-producing memory T cells present in the WT mice at any stage of
the response (starting from day 2), these data suggest it has to be the
indirect IL-4 pathway that suppresses the generation of specific Th1
cells in the WT mice, vs the IL-4-/- mice. The
indirect IL-4 pathway might also render the spleen a Th2-biasing organ,
vs peripheral LN. For example, Th2 immunity can be induced by injecting
Ags in IFA (21, 48, 49, 50, 51). The type 2 biasing effect is more
pronounced when the Ag is injected i.p. (when the priming occurs in the
spleen), as opposed to s.c. (when the priming occurs in LN)
(21). Thus, the indirect IL-4 pathway in the spleen might
explain the type 2 bias of the spleen.
The frequency of the cell type capable of the IL-4 bystander reaction
was comparable with the frequency of Ag-specific Th1 cells in the
spleen and with the frequency of cognate IL-4-producing Th2 cells
induced by immunizations with IFA and alum (21).
Therefore, in terms of creating type 1/type 2 environments in the
spleen, the magnitude of the indirect pathway IL-4 response is
considerable. Yet the frequency of the Ag-specific T cells and of the
IL-4 bystander cells is low, in the 1:5000 range. This low frequency
has made it difficult to directly phenotype the cell that produces the
bystander IL-4 in the spleen. The fact that the IL-4 bystander reaction
can be induced in RAG2-/- and SCID spleen cells
rules out as the cellular source B cells, T cells, and NK1.1 cells,
which require the rearrangement of their Ag receptors. Among the
recombinant cytokines tested, IL-3 was the most effective inducer of
IL-4 production in RAG2-/- spleen cells.
Basophils and mast cells are known to be regulated by IL-3
(52, 53, 54, 55, 56); therefore, this cell type seemed to be a prime
candidate for the IL-4 bystander reaction. Supporting this notion, two
different strains of mast cell-deficient mice showed a 3-fold reduced
IL-4 bystander response when their spleen cells were activated by rIL-3
(Fig. 3D) or by activated T cell culture supernatants (data
not shown). The residual IL-4-producing cells could either represent
immature mast cells that are present in these mice, or other cell types
capable of mediating the indirect IL-4 pathway.
In conclusion, we have shown that recall Ag-induced production of IL-4 in the spleen (and the blood) does not necessarily indicate the presence of Th2 cells. While the detection of recall Ag-induced IL-4 in mixed cell populations has been generally interpreted as evidence for Th2 or Th0 memory, our data show that this indirect pathway needs to be considered in immune diagnostics. The detection of such IL-4 does not reflect the long-term cytokine commitment of the T cell compartment that is of interest for providing insights, e.g., into the efficacy of vaccinations. Because this bystander reaction is radiation resistant, IL-4-/- APC or APC from organs that are void of the bystander cells (LN and thymus) should be used to reveal T cell-derived IL-4. Our identification of a distinct pathway of recall Ag-induced IL-4 production in certain organs, but not others, sheds light on partial type 2 immune response phenotypes, including the dissociated production of IL-4-dependent IgG1 and IgE. Further dissection of cognate vs indirect pathway IL-4 production will contribute to the better understanding of the mechanisms that underlie different facets of type 1 and type 2 immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul V. Lehmann, Department of Pathology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, BRB 929, Cleveland, OH 44106-4943. E-mail address: PVL2{at}po.cwru.edu
3 Abbreviations used in this paper: PLP, proteolipid protein; LN, lymph node; dLN, draining LN; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; PBST, PBS with Tween 20; WT, wild type.
Received for publication July 11, 2001. Accepted for publication October 15, 2001.
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