| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2% of human peripheral NK cells produced IL-13
protein in response to IL-2. Isolated NK cells from SCID and RAG-2
knockout (-/-) mice that lack T and B cells as well as normal mice
also can produce IL-13 mRNA and protein in response to IL-2. We
hypothesized that in the absence of IFN-
, IL-13-producing NK cells
may predominate in vivo. Utilizing IFN- knockout (-/-) mice as a
model system, IL-2-activated liver NK and T cells expressed 10-fold
more IL-13 and IL-5 mRNA and protein than normal controls following
IL-2 treatment in vitro. These results suggest that in the absence of
IFN-, an IL-13- and IL-5-producing NK and T cells predominate in
vivo. The existence of this cell type has important implications in
innate immunity given that the balance between IFN- and
IL-13/IL-5-producing NK cells may influence the early development of a
cell-mediated or humoral immune response. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, while Th2
cells were initially reported to produce IL-4, IL-5, IL-10, and also
IL-13. Th0 cells can produce a combination of the cytokines
characteristic of Th1 and Th2 cells, i.e., IL-2, IL-4, IL-13, IFN-,
and other cytokines (1, 2, 3, 4, 5). There is accumulating evidence
demonstrating that the failure to resolve infectious or inflammatory
diseases often results from an imbalance of these T helper cell subsets
rather than an insufficient immune response (6, 7).
NK cells are large granular lymphocytes that are derived from bone
marrow and display non-MHC-restricted cytotoxicity against a variety of
tumor, bone marrow-transplanted, allogenic target, and viral infected
cells (8). It is well known that NK cells are potent producers of
IFN- and granulocyte-macrophage-CSF
(GM-CSF)4 in response to
different extracellular signals. In particular, IL-1, IL-2, IL-12,
IL-18 (IFN--inducing factor), and TNF-
are thought to be potent
inducers or coinducers of IFN- or GM-CSF mRNA expression in NK cells
(9, 10, 11, 12, 13, 14, 15). Moreover, recent studies revealed that NK cells also were
potent producers of IL-5 and IL-10 (16, 17). It has been reported that
the majority of NK cells constitutively express an intermediate
affinity IL-2 receptor complex (IL-2R), which is composed of the ß
and common (c) subunits (18). IL-15 also is able to
activate NK cells via the ß and c subunits of IL-2R
(19, 20, 21).
In contrast to the positive effects such as IL-2, IL-12, and IL-15 on
NK function, IL-4 and IL-13 have been shown to inhibit NK gene
expression and function (5, 10). Recent studies in our laboratory have
shown that IL-4 and IL-13 can induce specific STAT6 protein DNA
complexes following treatment of NK cells with these ILs (22). IL-13
was initially described as a protein designated P600 and preferentially
produced by activated mouse Th2 cells. It has been shown that activated
CD4+, CD8+ T cells, EBV-transformed B cells,
and mast cells are able to express IL-13 (4, 5). However, IL-4 and
IL-13 production by NK cells in response to any stimulus has not been
previously demonstrated. IL-13 has multiple biological activities,
including up-regulation of CD23 and MHC class II expression on
monocytes and B cells (4, 5). Although IL-13 has only a low degree of
sequence homology with IL-4, IL-13 elicits B cell proliferation similar
to that induced by IL-4. For instance, IL-13 induces IgG4 and IgE
production by human B cells in vitro (4, 5, 23). Previous studies
showed that IL-13 like IL-4 also down-regulated some monocyte functions
including cytokine production (5). Furthermore, recent papers suggested
that IL-4 and IL-13 receptor complexes could share common components
IL-4R and IL-13R (5, 24).
In this report, we demonstrate that specific populations of NK cells
can produce significant levels of IL-13 mRNA and protein in response to
IL-2. In addition, in the absence of IFN- in vivo, a population of
NK cells that produces IL-13 and IL-5 in response to IL-2 can be
recruited into the liver and spleen, indicating that this population
may have a previously unknown influence on the development of
cell-mediated immunity. The potential importance of these findings in
the function of NK cells is discussed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant human (rh) IL-2 and rhIL-12 were obtained from
Hoffmann-La Roche (Nutley, NJ). Recombinant mouse IFN- was obtained
from Genentech (South San Francisco, CA).
Monoclonal antibodies
Peridinian chlorophyll protein-conjugated anti-human CD3
(Leu-4), phycoerythrin (PE)-anti-human CD8 (Leu-2a), anti-human
CD56 (Leu-19), FITC-conjugated anti-human CD4 (Leu-3a),
anti-human CD16 (Leu-11a), FITC-anti-human TCR-ß (WT31)
and TCR-
(TCR--1) mAbs were obtained from Becton Dickinson
(Mountain View, CA). PE-anti-mouse NK1.1 (PK136), PE-anti-mouse
DX5 (DX5), Cy-Chrome-anti-mouse CD3
(145–2C11),
FITC-anti-human IFN- (4S.B3, mouse IgG1), PE-anti-human
IL-13 mAbs (JES10–5A2, rat IgG1), or isotype-matched Ig were purchased
from PharMingen (San Diego, CA).
Cell lines
The NK3.3 human NK cell line has been previously described in detail (10). We have utilized a subline of NK3.3 (NKjp), and cultured the cells in RPMI 1640 supplemented with 15% heat-inactivated FBS, 10% Lymphocult-T (Biotest AG, Dreieich, Germany), 2 mM L-glutamine (BioWhittaker, Walkersville, MD), 100 U/ml penicillin, 100 µg/ml streptomycin (BioWhittaker) and 10 mM HEPES buffer (BioWhittaker).
Isolation of human NK cells
PBMC were isolated from buffy coats of leukophoresed normal, healthy volunteers by Ficoll-Hypaque. Adherent cells were removed by incubation on plastic dishes and nylon wool. Highly enriched populations of large granular lymphocytes (>95%) were obtained by centrifugation of nylon wool-passed cells on discontinuous density gradients of Percoll and anti-CD3 Ab depletion as previously described (9).
Establishment of human NK and T cell clones
NK and T cell clones were established from PBMC by a previously described method using H-medium (30% RPMI 1640 medium, 60% AIM-V medium (Life Technologies), 10% FBS containing 200 U/ml rhIL-2, 0.4 µg/ml PHA-P (Difco, Detroit, MI), and 0.8% human T-STIM with PHA (Becton Dickinson Labware, Bedford, MA)) (25, 26). NK and T cell clones were established by a limiting dilution method. Briefly, cells (at 0.2 cell/well) were cultured in the wells of 96-well, U-bottom plates (Falcon, Lincoln Park, NJ) with H-medium and irradiated allogenic PBMC from healthy donors. Afterward, NK or T cell clones were transferred to the wells of 24-well culture plates for further expansion with H-medium.
Mice
C57BL/6 (B6), SCID, and RAG-2 knockout (-/-) (B6 background)
(27) and IFN- (-/-) mice that contain a disrupted exon 2 of
IFN- genomic DNA (28) were used in this study. SCID and IFN-
knockout (-/-) mice were backcrossed for more than 10 generations
with B6 mice. These mice were maintained under specific pathogen-free
conditions and used for experiments at 8 to 12 wk of age.
Isolation of liver and splenic cells from IL-2-treated mice
Liver cell isolation.
NK cells were enriched from livers of IL-2-treated B6, SCID, RAG-2
(-/-), and IFN- (-/-) mice as follows. Mice were injected with
6 x 105 U rhIL-2 twice a day for 3 days
(36 x 105 U/total/mouse) to augment the number of
liver and spleen NK cells. On day 4, livers were perfused and
harvested. Single-cell suspensions of the livers were made using a
Stomacher 80 (Tekmer, Cincinnati, OH). The cell suspension was passed
through sterile gauze with HBSS, and cells were washed and resuspended
with HBSS. The cell suspension was passed through a cell strainer
(Becton Dickinson Labware, Franklin Lakes, NJ) with a 100-µm
pore size nylon filter and then washed with HBSS. Using Lympholyte-M
(Cedarlane Laboratories, Hornby, Ontario, Canada), mononuclear cells
were isolated by the density gradient centrifugation method. Isolated
cells were washed twice with HBSS and resuspended in RPMI 1640
containing 10% FBS for additional experiments. For further
purification, these cells were sorted by MoFlo (Cytomation, Fort
Collins, CO) using CyChrome-conjugated anti-mouse CD3 or
PE-anti-DX5 mAb as previously described (29).
Spleen cell isolation. Spleens were harvested from IL-2-treated mice as described above. Single-cell suspensions were made by passing the spleens through a metal mesh screen. RBC were lysed in water, and cells were washed and resuspended in RPMI 1640 containing 10% FBS. The cell suspension was then passed through a prewetted nylon wool column. Nylon wool-nonadherent cells were collected, washed, and resuspended in RPMI 1640 containing 10% FBS for additional experiments.
mRNA assays
Total RNA was isolated using a single-step phenol/chloroform extraction procedure (Trizol, Life Technologies). For Northern blotting analysis, 10 µg of total cellular RNA were analyzed by Northern blotting following electrophoresis on a 1% formaldehyde agarose gel, and hybridized with random-primed 32P-labeled cDNA probes as reported (12). For the RNase protection assay (RPA), 2.5–5 µg of total cytoplasmic RNA were analyzed using the RiboQuan kit (PharMingen) and [33P]UTP-labeled riboprobes as described by the manufacturer.
Cytokine assays
Human NK cell lines, clones, T cell clones, or mouse cells
were treated with different stimuli (as indicated in
Results) for the specified periods of time; cell-free
supernatants were collected and assayed for cytokine production by
ELISA. The specific ELISA kits utilized were: human IFN- (Biosource,
Fleurus, Belgium); human IL-4 (R&D Systems, Minneapolis, MN); human
IL-13 (R&D Systems); mouse IL-13 (R&D Systems); mouse IFN- (Genzyme,
Cambridge, MA); and mouse IL-5 (Endogen, Woburn, MA). The sensitivity
limits were 1.5, 5, 36, 1.5, 5, and 5 pg/ml, respectively.
Surface Ag and intracellular analysis by flow cytometry
Three-color analysis was performed using FACSort (Becton
Dickinson) as previously reported (29). Anti-mouse CD16/CD32 mAb
(2.4G2, PharMingen) was used to block the nonspecific bindings. To
detect the intracellular expression of cytokines, cultured cells were
stimulated with IL-2 or 10 ng/ml PMA (Sigma, St. Louis, MO) plus 1
µg/ml ionomycin (Sigma). To enhance intracellular protein secretion,
cells were treated with 2 µM monensin (Sigma) for 6 h at 37°C.
Following incubation, cells were treated with 20 µg/ml DNase I for 5
min at 37°C and washed twice with cold PBS. Then, cells were stained
by surface molecule-specific mAb (e.g., anti-CD3, anti-NK1.1)
as described before. These cells were fixed for 5 min at 37°C by the
addition of 4% paraformaldehyde-PBS to a final concentration of 1%,
centrifuged at 2600 rpm for 5 min, and then washed once with cold PBS
containing 0.1% BSA. The cells were permeabilized by a cold
permeabilization buffer (0.1% saponin (Sigma), 0.1% BSA, and 0.01 M
HEPES buffer containing PBS). The cells were incubated with
cytokine-specific mAb or isotype-matched mAb for 20 min on ice.
FITC-conjugated anti--smooth muscle actin (1A4, Sigma) was used
for a positive control. Following two washings with permeabilization
buffer, the intracellular expression was analyzed using a FACSort.
Thirty thousand cells were analyzed in each experiment.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our initial screening of cytokine production by the human NK 3.3
cell line was performed with a multiprobe RPA. Utilizing this assay, we
observed that in addition to IFN-, a protected RNA band
corresponding to IL-13 also was present (data not shown). Based on this
result, kinetics of IL-13, IFN-, and GM-CSF mRNA expression were
investigated by Northern blot analysis following treatment with IL-2
(100 U/ml) or a combination of PMA (10 ng/ml) and ionomycin (1
µg/ml). As shown in Fig. 1
, no IL-13 or
GM-CSF mRNA was expressed without stimulation, while IFN- mRNA was
constitutively expressed in NK3.3 as we had previously reported (12).
However, following stimulation, IL-13, IFN-, and GM-CSF mRNA were
detected at each time point (3 and 6 h) in response to IL-2 and
PMA plus ionomycin (Fig. 1). Moreover, the RPA revealed that IL-13 mRNA
was easily detectable 1 h after IL-2 stimulation (data not shown).
Interestingly, IL-13 mRNA expression in the NK 3.3 cell line 6 h
after IL-2 stimulation was much higher than that observed following
6 h of PMA/ionomycin stimulation (Fig. 1). These results are
opposite to what is seen for the IFN- and GM-CSF mRNAs and suggest
that IL-2 induces IL-13 by a pathway that is distinct from that
triggered by PMA plus ionomycin.
|
, significant levels of IL-13 and
IFN- protein were observed in the supernatants after IL-2 treatment.
Furthermore, the levels of IL-13 and IFN- protein correlated
directly with the concentration of IL-2 used in the stimulation (IL-2,
1 to 100 U/ml, Table I).
|
expression in both NK and T cells. Moreover, the combination of IL-2
and IL-12 strongly induces the development of IFN--producing NK and
T cells and synergistically increases IFN- production (30).
Therefore, we investigated whether the IL-13 production could be
modulated by IL-12 in NK3.3 cells. No significant enhancement of IL-13
protein production was observed in the response to IL-12 alone or IL-2
and IL-12 combinations, although IFN- protein was synergistically
increased in response to IL-2 and IL-12 (Table I
). Northern blotting
analysis also showed that no significant enhancement of IL-13 mRNA was
found in the response to IL-12 or to IL-2 and IL-12 (data not shown).
To investigate whether IL-13 and IFN--producing cells represented
distinct populations within the NK3.3 cells, we performed intracellular
cytokine staining. Representative two-color analysis of IL-2-activated
NK3.3 using FITC-conjugated mouse anti-human IFN- and
PE-conjugated rat anti-human IL-13 mAb is shown in Fig. 2. While no significant population of
cells expressing IL-13 and IFN- was found without stimulation (Fig. 2B), 50% of NK3.3 cells produced IL-13 and 4% of cells
expressed IFN- 18 h after IL-2 stimulation (100 U/ml) (Fig. 2C). The kinetics of IL-13 and IFN- also were
investigated at five different time points (0, 9, 18, 24, and 55
h). The percentage of IL-13-producing NK3.3 cells at each time point
were 0.5, 22.8, 49.8, 51.2, and 28.2%, respectively, while IFN- was
detected in 0.1, 3.6, 4.0, 5.4, and 5.4% of the population,
respectively. Therefore, the kinetics of IL-13 production by NK3.3
appeared to be different from that of IFN-. These results also are
similar to what is seen for the IL-13 and IFN- RNA kinetics in Fig. 1 and further support the hypothesis that the biochemical mechanisms
involved in the induction of these two cytokines may be different.
|
To analyze the production of IL-13 by human NK cells, we
established 21 NK clones from the purified human NK cells obtained from
3 donors by the limiting dilution method (25, 26). These clones were
all CD56+, CD16+, CD4-,
CD3-, TCRß-, and TCR-
(data not shown). A representative staining pattern of selected NK
clones is shown in Fig. 3. Although CD56
and CD16 expressions were brightly expressed on all of 21 NK clones as
shown in Fig. 3, CD8 was variably expressed on these clones. Seven
clones brightly expressed CD8, and 14 clones weakly expressed CD8 on
their surfaces. There was no significant change of the expression of
CD56, CD16, and CD8 molecules after prolonged culture of the clones
(data not shown).
|
. As shown in Table II, 6 of
12 NK clones produced detectable amounts of IL-13. Namely, three clones
(nk1, nk2, nk3) produced IL-13 in response to IL-2 and PMA + ionomycin,
and three clones (nk4, nk5, nk6) produced IL-13 in response to PMA plus
ionomycin alone. Interestingly, all the clones produced IFN- in
response to PMA plus ionomycin and four clones (nk1, nk2, nk3, and nk7)
produced significant amounts on IFN- in response to IL-2. These
results may suggest that other signals, as yet unidentified, might act
as a cofactor for IL-13 production by NK cells and synergize with IL-2.
Indeed, in contrast to the well-documented enhancement of IFN- and
GM-CSF in response to IL-2 and IL-12, no increase in IL-13 production
was observed in these NK in response to IL-12 alone or to IL-2 plus
IL-12 (data not shown). Thus, the effects of IL-12 appear to be on the
type 1 cytokine (IFN-, GM-CSF), and some other cytokine or chemokine
may synergize with IL-2 for the induction of IL-13. Three clones (nk1,
nk3, nk5) also produced significant levels of IL-4 in response to PMA
plus ionomycin. Similar results also were observed in human NK cell
lines. Although detectable levels of IL-4 protein were not observed, a
protected IL-4 mRNA band was observed in PMA/ionomycin-stimulated human
NK cell lines (NK3.3, NK92) by RPA (data not shown).
|
To determine the percentage of freshly isolated human peripheral
blood NK cells that were capable of producing IL-13, we analyzed total
NK cells obtained from three healthy donors. The purified NK cells were
CD3-CD16+CD56+, and the purity was
always >95%. The purified NK cells were resuspended in 10% FBS RPMI
1640 and stimulated with IL-2 (1000 U/ml) or PMA (10 ng/ml) plus
ionomycin (1 µg/ml) at 1 x 106/ml for 18 h.
IL-13-producing cells were then analyzed by intracellular cytokine
staining. Fig. 2F shows a representative staining pattern of
IL-2-activated peripheral NK cells. Approximately 2–3% of the
peripheral NK cells were found to produce IL-13 after 18 h of IL-2
or PMA plus ionomycin stimulation (data not shown) in the three donors.
This low percentage suggests that IL-13-producing NK cells represent a
minor population of the normal NK population in the periphery.
IL-13-producing NK cells in the C57BL/6, SCID, and RAG-2 knockout mice
In the mouse liver and spleen cells, NK cells are 10%
and 1–2% of the lymphocytes in these organs, respectively (31).
Therefore, we used the phenomenon of IL-2 rebound effects to generate
large quantities of murine NK cells in the liver and spleen (31) to
investigate if mouse NK cells can produce IL-13. Repeated experiments
showed that after generation of NK cells in vivo with IL-2,
5–8 x 106 lymphocytes/liver and 7–12 x
107 lymphocytes/spleen were isolated from IL-2-treated B6
mice. As shown in Table III, the
percentage of CD3-NK1.1+ NK cells in liver and
spleen was 50–80% and 5–20%, respectively. Then enriched liver
and spleen NK cells were analyzed for RNA expression in response to
IL-2. Significant amounts of IL-13 and IL-5 mRNA and proteins were
found in the cells from B6 mice (Table III, Fig. 4). Highly purified liver NK cells also
produced IL-13 protein in response to IL-2 (Table IV).
|
|
|
3–5 x 107 lymphocytes/spleen were isolated from
in vivo IL-2-treated RAG-2 (-/-) mice. The percentage of
CD3-NK1.1+ NK cells in liver and spleen was
90–95% and 80–90%, respectively. There was no significant
population of CD3+CD4+ or
CD3+CD8+ T cell subsets (Table III and data not
shown). The liver and spleen NK cells from RAG-2 (-/-) mice produced
small amounts of IL-13 and IL-5 but produced greater amounts of IFN-
in response to IL-2 (Table III). Interestingly, the NK cells isolated
from the liver produced more IL-13 than the corresponding population
isolated from the spleen (Table III). Moreover, isolated liver NK cells
from SCID mice, which also lack most T cells, expressed IL-13 and IL-5
mRNA and protein as analyzed by RPA (data not shown) and ELISA (Table III). These results suggest that in RAG-2 (-/-) mice, SCID mice, and
normal mice, IL-13/IL-5-producing NK cells represents a subpopulation
of the total NK cells.
IL-13-producing NK cells in the IFN- knockout mouse
To investigate whether or not these cells may play an important
physiological role in vivo, we hypothesized that in the absence of
IFN-, substantially more of the IL-13-producing NK cells may
predominate. To test this hypothesis, we used the phenomenon of IL-2
rebound effects to generate large quantities of murine NK cells in the
liver and spleen from B6 and IFN- (-/-) mice. Our rationale for
this experiment was that IFN- may act to suppress the
IL-13-producing population of NK cells and that in the absence of
IFN-, this population would predominate. As shown in Table III,
CD3-NK1.1+ NK cells in the spleen and liver
were accumulated from both IFN- (-/-) and normal mice.
Approximately 5–8 x 106 lymphocytes per liver and
7–10 x 107 lymphocytes/spleen were isolated from
IL-2-treated IFN- (-/-) mice. Then spleen and liver cells were
analyzed for RNA and protein production in response to IL-2 treatment
in vitro. As shown in Fig. 4, higher levels of IL-13 and IL-5 mRNA were
observed in the cells obtained from IFN- knockout mice than that
obtained from normal animals (C57BL/6). It is of interest that the
IFN- mRNA and protein seen in normal animals was not reinduced in
vitro by IL-2, in contrast to the IL-13 and IL-5 (Table III, Fig. 4).
This is consistent with the results observed with the human NK clones
given that most clones would not express significant IFN- in
response to IL-2, although the clones were established in the presence
of IL-2. The IFN- mRNA-protected bands seen in the knockout mice may
be due to readthrough of the mRNA since the IFN- riboprobe spans
exons 3 and 4 of the murine IFN- genomic DNA. In a repeat
experiment, identical results for IL-13 and IL-5 were obtained after
only 2 h of IL-2 treatment (100 U/ml) in vitro (data not shown).
As shown in Table III, in IFN- (-/-) mice, IL-2-activated liver
and spleen cells produced 10–60-fold more IL-13 and IL-5 protein
than seen in B6 mice, and there was no IFN- production. Moreover,
IL-13 and IL-5 protein production correlated with the percentage of
NK1.1+CD3- cells in both the spleen and liver
in the IFN- knockout mice. These results suggest that in IFN-
(-/-) mice, CD3-NK1.1+ NK cells can produce
much more IL-13 and IL-5 than seen in normal mice.
We further purified liver NK cells to verify whether highly purified NK
cells in IFN- knockout mice can produce IL-13 by depletion of
CD3+ T cells. Liver cells were isolated from IFN-
knockout and C57BL/6 (B6) mice and were sorted using anti-CD3 mAbs
to obtain highly purified CD3- NK cells and
DX5+ NK cells. Representative results are shown in Table IV. In B6 mice, IL-2-stimulated CD3- NK cells and
CD3+ T cells produced small amounts of IL-13 (20 and 26
pg/ml, respectively). We then sorted liver cells from IFN- (-/-)
mice using anti-DX5 and anti-CD3 mAbs and isolated purified
DX5+ NK cells and DX5- T cells.
DX5+ NK cells, CD3- NK cells,
DX5- T cells, and CD3+ T cells produced IL-13
(66, 222, 361, and 2686 pg/ml, respectively). Greater amounts of IL-13
were observed in the supernatant of IL-2-activated highly purified
liver NK and T cells than seen in normal controls. These results
suggest that: 1) liver T cells from IFN- (-/-) mice also can
produce more IL-13 than in normal controls following IL-2 treatment in
vivo; 2) cross-linking of CD3 by CD3 mAb (2C11) can up-regulate IL-13
production in CD3+ T cells; and 3) depletion of T cells
from the population may result in decreased IL-13 production by NK
cells. These results suggest that T cell-NK cell interaction might be
important for optimal IL-13 production in the IFN- knockout mice.
This issue is now under investigation.
We wished to determine whether IFN- could act to inhibit the
replication or directly block IL-13 production by the IL-13-producing
NK cells. After generation of the cells in vivo with IL-2, isolated
liver cells were incubated overnight with no stimulation, rhIL-2 (100
U/ml), murine rIFN- (10, 100, 1000 U/ml) or a combination of IL-2
plus IFN-. Although exogenous IFN- partially inhibited
(20–40%) IL-13 and IL-5 production, IFN- did not directly block
the IL-2-induced expression of IL-13 or IL-5 by NK cells in IFN-
(-/-) mice (data not shown). Thus, it is not yet clear if IFN-
itself may modulate the levels of IL-13-producing NK cells or if it may
act at an earlier precursor stage.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2–3% of peripheral NK cells expressed IL-13
protein in response to IL-2 or PMA plus ionomycin. Thus, this
population of NK cells represents a subpopulation of the total
circulating NK cells.
The physiological relevance of IL-13 production in NK cells is an
important question given that cell lines and individual clones may not
be truly representative models. To verify this hypothesis in a
situation in vivo where IL-13-producing NK cells may play an important
role, we used the phenomenon of IL-2 rebound and knockout mice as model
systems to investigate whether an IL-13-producing NK subset may be
present in vivo. We showed in this study that by treating mice for 3
days with rhIL-2, large numbers of NK cells migrate to both the spleen
and liver in SCID, RAG-2 (-/-), IFN- (-/-), and normal mice. We
hypothesized that in the absence of IFN-, there would be no
environmental pressure against the predominance of an IL-13-producing
NK population. We found that in the IFN- knockout mice, NK cells in
liver and spleen cells could produce 10-fold more IL-13 and IL-5 mRNA
and protein than seen in control mice when the cells were retreated in
vitro. Moreover, in IFN- knockout mice, IL-2-activated liver T cell
subsets also could produce greater amounts of IL-13 than seen in normal
mice.
Thus, these cells represent a novel NK population, not predominant in spleen or liver in normal mice. Interestingly, these cells could not be distinguished from the NK cells obtained from normal mice when analyzed for expression of different Ly-49 receptor families (35), Ly-49A, C, D, and G2 (data not shown). Experiments are now under way to determine whether unique cell surface molecules may exist on the IL-13-producing NK cells.
IL-4 and IL-13 is thought to be critical for the differentiation of
naive T cells into Th2 T cells (5, 6, 7). The IL-4/IL-13-producing Th2 T
cells in vivo is thought to represent a small subpopulation of the
total T cell subsets (4, 5). Recent studies have demonstrated that
NK1.1+ T cell subsets can be a stronger producer of IL-4
than conventional T cell subsets (32, 33, 34). In this study, we showed
that in IFN- knockout mice, IL-2-activated purified T cells produced
10–100-fold more IL-13 protein than normal mice. These results suggest
that in the absence of IFN-, Th2 cytokine production by both T and
NK cells may predominate in vivo. We speculate that IFN- may
suppress the development of IL-13-producing T and NK cells in vivo and
induce a Th1 cytokine profile.
In the normal mouse, NK cells are a minor population of total
lymphocytes; spleen and liver NK cells are 10% and 1 to 2% of the
total lymphocytes, respectively (31). A subpopulation of mouse T cells
(NK-T) has an NK marker (NK1.1) on the cell surface (32, 33). Thus,
analysis of pure NK cells is difficult due to NK-T cell contamination.
In this study, we also have utilized RAG-2 knockout mice that lack T
and B cells (27) and the phenomenon of IL-2 rebound to evaluate the NK
cells in the absence of any T cells or B cells. We demonstrate that
isolated NK cells from RAG-2 (-/-) mice produce IL-13 in response to
IL-2. Interestingly, the liver NK cells isolated from SCID and RAG-2
(-/-) mice produced more IL-13 and IL-5 than the corresponding cells
isolated from the spleen. Moreover, both liver and spleen NK cells in
SCID and RAG-2 (-/-) mice expressed relatively high levels of NK1.1,
DX5, and CD25 on the cell surface (data not shown). These results
suggest that the NK cells that migrated into the liver and spleen
following IL-2 treatment in vivo might represent different NK
subpopulations. Consistent with this hypothesis, our previous study
revealed that the majority of accumulated liver NK cells in biological
response modifier-treated mice could be derived from bone marrow but
not from spleen (36).
NK cells have been reported to be a potent producers of IFN-,
GM-CSF, TNF-, IL-5, and IL-10 in response to different stimuli
(9, 10, 11, 12, 13, 14, 15, 16, 17). However, to our best knowledge, there have been no reports
demonstrating that NK cells can produce IL-13 and/or IL-4. It has been
shown that activated human CD4+CD8+ Th0, Th1,
and Th2 cells, EBV-transformed B cell, and mast cells are able to
express IL-13 (4, 5). IL-13 as well as IL-4 is involved in the
development of humoral immunity (5, 6, 7). IL-13 and IL-4 also affect B
cell and monocyte function. As seen with IL-4, IL-13 induces production
of IgM, IgG4 and IgE production by human B cells when the appropriate
second signal (CD40 ligand (CD40L)-CD40 interaction) is provided (4, 5). Murine and human CD40L had been cloned and found to be membrane
glycoprotein on activated T cells (37). Recently, mast cell,
eosinophils, and B cells also can express functional CD40L on their
surfaces. The CD40L-CD40 interaction has been demonstrated to be
necessary for T cell-dependent B cell activation and maturation
(37, 38, 39). It has been suggested that NK cells play a role in B cell
differentiation and Ig production (40). A recent study has revealed
that IL-2-activated human NK cells expressed functional CD40L on their
surfaces (41). Taken together, that fact that activated NK cells can
produce IL-13 and express CD40L may account for their role in inducing
B cell differentiation and Ig production.
It has been well known that NK cells can produce IFN- in response to
IL-2 and IL-12 (30). In this study, the human NK cell line, NK3.3,
produced significant levels of IFN- in response to a relatively low
dose of IL-2 (10 U/ml). However, many of human NK clones would not
express IFN- in response to high dose of IL-2 (1000 U/ml), although
most of T cell clones tested produced IFN- in response to IL-2 (data
not shown). In the NK clone nk1, the response to IL-2 after long term
culture (more than 10 wk) was much better than in initial culture
conditions (data not shown). Moreover, IFN- mRNA seen in the NK
cells isolated from normal mice following IL-2 treatment in vivo was
not reinduced in vitro by IL-2. Thus, chronic exposure of the cells to
IL-2 might result in a type of anergy with respect to gene induction.
These results suggest that NK cell lines, T cell clones, and a long
term-cultured NK clone and line may express high affinity IL-2R (,
ß, and c chains), while most of NK clones and NK cells
in normal mice may express intermediate IL-2R (ß and c
chains). FACS analysis revealed that most (>90%) NK cells in human
peripheral blood and in normal mice did not express IL-2R chain
(CD25) on their surfaces (data not shown) as previously reported (18, 42). It will be important to analyze the transcription factors
activated in these different cell populations to determine how the
IL-2-signaling pathways differ in these cell populations.
In preliminary experiments, we have found that in normal mice a
significant percentage of the lymphocytes underwent apoptosis even in
the presence of IL-2, and IFN- treatment did not affect this
percentage. In the IFN- (-/-) mice, a similar percentage of
cells underwent apoptosis in the absence or in the presence of IFN-.
However, in sharp contrast to the results observed with the normal
mice, IL-2 protected the cells obtained from the knockout mice
from apoptosis. This result suggests that these cells are distinct from
the normal population of NK cells in their response to IL-2 at both the
molecular and cellular levels. Whether or not IFN- affects the early
progenitors of these cells or induces a factor that suppresses their
proliferation is under investigation.
The absence of bcl-2 expression in bcl-2-deficient mice results in a complete disappearance of lymphocytes by 6 wk of postnatal life due to massive apoptosis (43, 44). bcl-2 can suppress induction of apoptosis by various cytotoxic treatments (19, 45). Some bcl-2-related genes, such as bcl-XL, ced-9, and BHRF-1 also can exert a bcl-2-like antiapoptotic effect (45). Previous studies showed that IL-2 could increase bcl-2 expression and prevent apoptosis in activated T cells (45). Therefore, bcl-2 is thought to be important for sustaining the survival of lymphocytes (19). We then investigated whether IL-2 could up-regulate bcl-2and/or bcl-XL and thus explain the differential effects we have observed. However, there was no difference in either bcl-2 or bcl-XL mRNA expression in the cells obtained from both control and knockout mice (data not shown), thus indicating that other mechanisms may be involved in the effects of IL-2 on apoptosis.
Given our observation that IFN- protein did not induce apoptosis in
the cells obtained from the IFN- knockout mice or directly block the
IL-2-induced expression of IL-13, it is unclear as to whether or not
IFN- directly influences this IL-13-producing NK and T cell
subset. Therefore, one possibility is that IFN- suppresses
the overall initial development of these cells in the bone marrow and
that in the absence of IFN-, these cells predominate. Furthermore,
it is possible that once these cells mature, they may lack IFN-
receptors and become refractory to the effects of IFN-. A previous
study showed that IFN- inhibited the development of Th2 clones
producing IL-4 (46). Alternatively, IFN- may suppress the production
of a growth factor needed for the growth of these cells. These
hypotheses warrant further analysis.
Many lines of study have reported localization of Th1- and Th2-type T cells in vivo. For instance, CD4+ Th1 cells were predominant in tuberculoid lesions induced by Mycobacterium leprae, in human and animal models of insulin-dependent diabetes mellitus, and also in the affected organs of sarcoidosis (6, 7, 25). IL-4-producing Th2-type CD4+ T cells were observed in the bronchoalveolar regions of patients with atopic asthma, while CD8+ T cells from lepromatous lesions also produced Th2-type cytokines. Based on our results, we would speculate that this NK subset might selectively localize at specific sites in the body, depending on the specific inflammatory trigger. For instance, the IL-13-producing NK subset may play a previously unrecognized role in pathogenesis of patients with atopic asthma and lepromatous as the predominance of Th2-type T cells in these conditions may affect or be affected by IL-13/IL-5-producing NK cells.
In conclusion, this study has demonstrated IL-13 production by human
and mouse NK cells in response to IL-2. Using IFN- (-/-) mice as a
model system, IL-2-activated NK and also T cells in liver and spleen
cells could produce 10-fold more IL-13 mRNA and protein than seen in
control B6 mice. Our results suggest that the absence of IFN- may
favor a Th2 cytokine expression profile in both NK and T cells. Mosmann
and colleagues have classified Th subsets as their cytokine productions
(1, 2, 5). Namely, Th1 cells mainly produce IL-2 and IFN-; Th2 cells
produce IL-4, IL-5, IL-6, IL-10, and also IL-13. Collectively, our
findings revealed that NK cells could be strong producers of Th2-type
cytokines in the absence of IFN-.
It will be of particular interest to determine the role of these cells in both cell-mediated and humoral immune responses.
|
Acknowledgments |
|---|
Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (47).
|
Footnotes |
|---|
2 Recipient of the 1997 Fukuoka Cancer Society Award (Fukuoka, Japan).
3 Address correspondence and reprint requests to Dr. Howard A. Young, Laboratory of Experimental Immunology, Division of Basic Sciences, Biological Response Modifiers Program, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address:
4 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage-colony-stimulating factor; c subunit, common subunit; RPA, ribonuclease protection assay; rh, recombinant human; PE, phycoerythrin; CD40L, CD40 ligand.
Received for publication July 7, 1998. Accepted for publication September 1, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by human large granular lymphocytes. J. Immunol. 139:724.[Abstract]
, interleukin-1ß and interferon--inducing factor on the production of interferon- by natural killer. Eur. J. Immunol. 27:2787.[Medline]
production induced by IL-2 and IL-12 in a human NK cell line. J. Leukoc. Biol. 58:225.[Abstract]
induction by natural killer cell stimulatory factor (NKSF/IL-12): role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2. J. Immunol. 148:92.[Abstract]
production by T cells. Nature 378:88.[Medline]
chain with the IL-2Rß chain in functional intermediate-affinity IL-2R. J. Exp. Med. 176:531. genes. Science 259:1739.ß in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc. Natl. Acad. Sci. USA 91:3700. in immune regulation. I. IFN- inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140:4245.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  M. R. Hepworth and R. K. Grencis Disruption of Th2 Immunity Results in a Gender-Specific Expansion of IL-13 Producing Accessory NK Cells during Helminth Infection J. Immunol., September 15, 2009; 183(6): 3906 - 3914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Iannello, O. Debbeche, S. Samarani, and A. Ahmad Antiviral NK cell responses in HIV infection: II. viral strategies for evasion and lessons for immunotherapy and vaccination J. Leukoc. Biol., July 1, 2008; 84(1): 27 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Feng, M. Kaviratne, A. G. Rothfuchs, A. Cheever, S. Hieny, H. A. Young, T. A. Wynn, and A. Sher NK Cell-Derived IFN-{gamma} Differentially Regulates Innate Resistance and Neutrophil Response in T Cell-Deficient Hosts Infected with Mycobacterium tuberculosis J. Immunol., November 15, 2006; 177(10): 7086 - 7093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Gao, P. Schwartzberg, J. A. Wilder, B. R. Blazar, and D. Yuan B Cell Induction of IL-13 Expression in NK Cells: Role of CD244 and SLAM-Associated Protein. J. Immunol., March 1, 2006; 176(5): 2758 - 2764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Taube, N. Miyahara, V. Ott, B. Swanson, K. Takeda, J. Loader, L. D. Shultz, A. M. Tager, A. D. Luster, A. Dakhama, et al. The Leukotriene B4 Receptor (BLT1) Is Required for Effector CD8+ T Cell-Mediated, Mast Cell-Dependent Airway Hyperresponsiveness. J. Immunol., March 1, 2006; 176(5): 3157 - 3164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-M. Kang, A.-S. Jang, M.-H. Ahn, J.-A. Shin, J.-H. Kim, Y.-S. Choi, T.-Y. Rhim, and C.-S. Park Interleukin-25 and Interleukin-13 Production by Alveolar Macrophages in Response to Particles Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 290 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. McDermott, N. E. Humphreys, S. P. Forman, D. D. Donaldson, and R. K. Grencis Intraepithelial NK Cell-Derived IL-13 Induces Intestinal Pathology Associated with Nematode Infection J. Immunol., September 1, 2005; 175(5): 3207 - 3213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miyahara, K. Takeda, S. Miyahara, S. Matsubara, T. Koya, A. Joetham, E. Krishnan, A. Dakhama, B. Haribabu, and E. W. Gelfand Requirement for Leukotriene B4 Receptor 1 in Allergen-induced Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 161 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Gao, T. Dang, W. A. Dunnick, J. T. Collins, B. R. Blazar, and D. Yuan Receptors and Counterreceptors Involved in NK-B Cell Interactions J. Immunol., April 1, 2005; 174(7): 4113 - 4119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Rodriguez-Galan, J. H. Bream, A. Farr, and H. A. Young Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression J. Immunol., March 1, 2005; 174(5): 2796 - 2804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Katsumoto, M. Kimura, M. Yamashita, H. Hosokawa, K. Hashimoto, A. Hasegawa, M. Omori, T. Miyamoto, M. Taniguchi, and T. Nakayama STAT6-Dependent Differentiation and Production of IL-5 and IL-13 in Murine NK2 Cells J. Immunol., October 15, 2004; 173(8): 4967 - 4975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Taube, X. Wei, C. H. Swasey, A. Joetham, S. Zarini, T. Lively, K. Takeda, J. Loader, N. Miyahara, T. Kodama, et al. Mast Cells, Fc{epsilon}RI, and IL-13 Are Required for Development of Airway Hyperresponsiveness after Aerosolized Allergen Exposure in the Absence of Adjuvant J. Immunol., May 15, 2004; 172(10): 6398 - 6406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Yuan, R. Bibi, and T. Dang The role of adjuvant on the regulatory effects of NK cells on B cell responses as revealed by a new model of NK cell deficiency Int. Immunol., May 1, 2004; 16(5): 707 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Miyahara, K. Takeda, T. Kodama, A. Joetham, C. Taube, J.-W. Park, S. Miyahara, A. Balhorn, A. Dakhama, and E. W. Gelfand Contribution of Antigen-Primed CD8+ T Cells to the Development of Airway Hyperresponsiveness and Inflammation Is Associated with IL-13 J. Immunol., February 15, 2004; 172(4): 2549 - 2558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Loza and B. Perussia Differential regulation of NK cell proliferation by type I and type II IFN Int. Immunol., January 1, 2004; 16(1): 23 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Azam, D. Novick, P. Bufler, D.-Y. Yoon, M. Rubinstein, C. A. Dinarello, and S. H. Kim Identification of a Critical Ig-Like Domain in IL-18 Receptor {alpha} and Characterization of a Functional IL-18 Receptor Complex J. Immunol., December 15, 2003; 171(12): 6574 - 6580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshino, H. Nakamura, M. Okamoto, S. Kato, S. Araya, K. Nomiyama, K. Oizumi, H. A. Young, H. Aizawa, and J. Yodoi Redox-active Protein Thioredoxin Prevents Proinflammatory Cytokine- or Bleomycin-induced Lung Injury Am. J. Respir. Crit. Care Med., November 1, 2003; 168(9): 1075 - 1083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Bream, R. E. Curiel, C.-R. Yu, C. E. Egwuagu, M. J. Grusby, T. M. Aune, and H. A. Young IL-4 synergistically enhances both IL-2- and IL-12-induced IFN-{gamma} expression in murine NK cells Blood, July 1, 2003; 102(1): 207 - 214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Whittaker, N. Niu, U.-A. Temann, A. Stoddard, R. A. Flavell, A. Ray, R. J. Homer, and L. Cohn Interleukin-13 Mediates a Fundamental Pathway for Airway Epithelial Mucus Induced by CD4 T Cells and Interleukin-9 Am. J. Respir. Cell Mol. Biol., November 1, 2002; 27(5): 593 - 602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Korten, L. Volkmann, M. Saeftel, K. Fischer, M. Taniguchi, B. Fleischer, and A. Hoerauf Expansion of NK Cells with Reduction of Their Inhibitory Ly-49A, Ly-49C, and Ly-49G2 Receptor-Expressing Subsets in a Murine Helminth Infection: Contribution to Parasite Control J. Immunol., May 15, 2002; 168(10): 5199 - 5206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Kim, T. Azam, D. Novick, D.-Y. Yoon, L. L. Reznikov, P. Bufler, M. Rubinstein, and C. A. Dinarello Identification of Amino Acid Residues Critical for Biological Activity in Human Interleukin-18 J. Biol. Chem., March 22, 2002; 277(13): 10998 - 11003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Shimozato, J. R. Ortaldo, K. L. Komschlies, and H. A. Young Impaired NK Cell Development in an IFN-{gamma} Transgenic Mouse: Aberrantly Expressed IFN-{gamma} Enhances Hematopoietic Stem Cell Apoptosis and Affects NK Cell Differentiation J. Immunol., February 15, 2002; 168(4): 1746 - 1752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Loza, L. Zamai, L. Azzoni, E. Rosati, and B. Perussia Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells Blood, February 15, 2002; 99(4): 1273 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Okamoto, S. Kato, K. Oizumi, M. Kinoshita, Y. Inoue, K. Hoshino, S. Akira, A. N. J. Mckenzie, H. A. Young, and T. Hoshino Interleukin 18 (IL-18) in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia Blood, February 15, 2002; 99(4): 1289 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-J. Lee, Y.-S. Cho, M.-C. Cho, J.-H. Shim, K.-A. Lee, K.-K. Ko, Y. K. Choe, S.-N. Park, T. Hoshino, S. Kim, et al. Both E6 and E7 Oncoproteins of Human Papillomavirus 16 Inhibit IL-18-Induced IFN-{{gamma}} Production in Human Peripheral Blood Mononuclear and NK Cells J. Immunol., July 1, 2001; 167(1): 497 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshino, Y. Kawase, M. Okamoto, K. Yokota, K. Yoshino, K.-i. Yamamura, J.-i. Miyazaki, H. A. Young, and K. Oizumi Cutting Edge: IL-18-Transgenic Mice: In Vivo Evidence of a Broad Role for IL-18 in Modulating Immune Function J. Immunol., June 15, 2001; 166(12): 7014 - 7018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Sayers, A. D. Brooks, J. M. Ward, T. Hoshino, W. E. Bere, G. W. Wiegand, J. M. Kelley, and M. J. Smyth The Restricted Expression of Granzyme M in Human Lymphocytes J. Immunol., January 15, 2001; 166(2): 765 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Matsukawa, C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff, R. M. Strieter, and S. L. Kunkel Expression and Contribution of Endogenous IL-13 in an Experimental Model of Sepsis J. Immunol., March 1, 2000; 164(5): 2738 - 2744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C. Pien and C. A. Biron Compartmental Differences in NK Cell Responsiveness to IL-12 During Lymphocytic Choriomeningitis Virus Infection J. Immunol., January 15, 2000; 164(2): 994 - 1001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. Smeltz, N. A. Wolf, and R. H. Swanborg Inhibition of Autoimmune T Cell Responses in the DA Rat by Bone Marrow-Derived NK Cells In Vitro: Implications for Autoimmunity J. Immunol., August 1, 1999; 163(3): 1390 - 1397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshino, R. H. Wiltrout, and H. A. Young IL-18 Is a Potent Coinducer of IL-13 in NK and T Cells: A New Potential Role for IL-18 in Modulating the Immune Response J. Immunol., May 1, 1999; 162(9): 5070 - 5077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I.-C. HO, J.I. KIM, S.J. SZABO, and L.H. GLIMCHER Tissue-specific Regulation of Cytokine Gene Expression Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 573 - 584. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
