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Departments of
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Microbiology,
Surgery,
Pathology and Laboratory Medicine, and
§
Hematology and Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ and
TCR
ß+CD8+ iIELs are reduced, and in
lethally irradiated SPF IL-2+/+ mice, reconstituted with
IL-2-/- bone marrow TCR+ iIELs fail to
develop, consistent with an important role of IL-2/IL-2R signaling in
the development of iIELs. Consequently, our findings demonstrate
that the colitis seen in SPF IL-2-/- mice depends upon
the presence of intestinal bacterial flora and that environmental Ags
are not responsible for the anemia and extraintestinal lymphoid
hyperplasia that occur in IL-2-/- mice. Thus, germfree
IL-2-/- mice represent a unique system in which the role
of IL-2 deficiency in hemopoietic and immune system disorders can be
investigated in dissociation from complications that may arise due to
colitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hypothesis that disease induction is caused by a breakdown in the
regulation of peripheral T cells is supported by several observations.
First, IL-2 has been demonstrated to mediate the termination of clonal
expansion by inducing apoptosis (6). Second, superantigen-stimulated T
cells from IL-2-/- (7) and IL-2R -chain-deficient (8)
mice are more resistant to Fas-induced apoptosis than those from
wild-type mice. Finally, the onset of the disease in
IL-2-/- mice reared and maintained in a specific
pathogen-free (SPF) environment is delayed (1). Together, these
observations suggest that disease is caused by dysregulated peripheral
T cell responses to environmental Ags.
Despite the initial analysis of IL-2-/- mice indicating normal T cell development (9), the recent finding that IL-2-/- athymic mice fail to develop autoimmune or lymphoproliferative disorders demonstrates that disease initiation depends upon the intrathymic differentiation of IL-2-/- T cells (10). Attempts to identify abnormalities in intrathymic T cell development and negative selection in IL-2-/- mice, however, have produced contradictory results. The marked skewing toward the CD4-8+ phenotype in the thymi of nonmanipulated IL-2-/- mice (11) and the decreased cortical apoptosis and loss of immature CD4+8+ (double-positive) and accumulation of CD4+ and CD8+ (single-positive) thymocytes in IL-2-/- mice following antigenic stimulation (12) are consistent with abnormal intrathymic development. However, the normal selection and deletion of thymocyte subsets by endogenous superantigens or following chronic administration of Ag to MHC class I-restricted TCR-transgenic mice in the absence of IL-2 (13) argue for T cell selection mechanisms being intact in these mice.
An obligatory role for IL-2 in the normal development of at least some
T cell populations is supported by the recent finding that the
composition of the intestinal intraepithelial lymphocytes (iIEL)
compartment is greatly altered in IL-2R ß-chain-deficient mice (14).
In particular, there is a marked decrease in
TCRß+CD8+ and TCR+
iIEL subsets. However, since both IL-2 and IL-15 signal through the
IL-2R ß-chain (15, 16), it is difficult to distinguish the cytokine
that is responsible for the iIEL developmental defect in these
mice.
To investigate further the mechanism of disease in
IL-2-/- mice, gnotobiotic (germfree)
IL-2-/- mice were rederived and evaluated for signs of
hemopoietic and immune disorders. The composition of the iIEL
repertoire was also evaluated in these animals. The results show that
germfree IL-2-/- mice develop hemopoietic disorders
similar to those in SPF IL-2-/- mice, consistent with the
hypothesis that defective central and/or peripheral tolerance to self
Ags results in autoimmunity. The absence of colitis and abnormalities
in the development of TCR+ iIEL subsets in germfree
IL-2-/- mice and IL-2-/- bone marrow
chimeras also demonstrate that intestinal lesions in SPF
IL-2-/- mice may arise as a consequence of an abnormal
mucosal immune response to enteric Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gnotobiotic (germfree) IL-2-/- mice (Taconic Farms, Germantown, NY), backcrossed eight generations onto the C57BL.6 background, were housed in isolator cages within a flexible film isolator in the gnotobiotic facility of the Biology Department, University of Pennsylvania. The germfree status of IL-2+/+, IL-2+/-, and IL-2-/- animals was verified by bacteriologic, histologic, and serologic analysis of tissues and fluids at autopsy. IL-2-/- mice obtained from heterozygous matings were identified from samples of tail DNA using a previously described PCR-based method (9). Heterozygote and/or wild-type littermates were used as controls. B6.SJL-PtprcaPep3b/BoyJ(Ly5a) (B6(CD45.1)) mice were a gift from Dr. Edward Scott (University of Pennsylvania, Philadelphia, PA).
Histopathology
Tissues removed at autopsy were fixed in formalin and embedded in paraffin. Sections were stained according to standard protocols with hematoxylin-eosin or stains for the detection of microorganisms that included Gram, acid-fast, Warthin-Starry silver, and Gomori methanamine-silver nitrate stains.
Cell preparation
Blood was obtained by tail bleeding or cardiac puncture and was collected in polypropylene tubes containing heparin. Blood counts were obtained using a Baker 1000 automated cell counter (Coulter, Hialeah, FL) and leukocyte differential counts were performed manually on Wright-stained blood smears. Mononuclear cells from thymus and spleen were prepared by disrupting intact tissues into ice-cold PBS. Bone marrow cells were obtained from femurs and tibias by flushing the bones with PBS and scraping the bone fragments. To prepare iIEL, the small intestine was opened longitudinally in HBSS, Peyer’s patches were removed, and the intestine was cut into small fragments that were incubated in HBSS containing 0.1 M EDTA and 5 mM DTT in an Erlenmeyer flask in a shaking water bath at 37°C for 30 min. Filtered cells were enriched for lymphocytes by Percoll gradient centrifugation, removing cells between the 65 and 45% interface. The purity of the iIEL was assessed by the degree of contamination by lamina propria-derived B220+, CD3- B cells. Intrahepatic lymphocytes were isolated by first irrigating the intact tissue with PBS to remove circulating cells. The tissue was then disrupted by passing it through a steel mesh, and the lymphocytes were enriched by Percoll gradient centrifugation as described above.
Bone marrow chimeras
B6(CD45.1) mice (n = 4–5/group) were lethally irradiated (1.1 Gy) and injected with 1 x 106 unmanipulated bone marrow cells from B6(CD45.1) syngeneic donors or IL-2-/- (CD45.2) congenic donors. Control mice were lethally irradiated and injected with an equal volume of PBS. Control mice died 10 to 14 days postirradiation. Experimental mice were euthanized at 90 days postirradiation, and iIELs were isolated as described.
Abs and flow cytometry
The following mouse and rat mAbs were used to stain and analyze
thymocytes isolated from germfree IL-2-/- and
IL-2+/+ mice: CD90/Thy 1.2 (30H.12; Life Technologies,
Gaithersburg, MD), CD3 (29B; Life Technologies), CD4 (CT-CD4; Caltag
Laboratories, San Francisco, CA), CD8 (53-6.7; Life Technologies),
CD8ß (CT-CD8b; Caltag Laboratories), CD11b/Mac-1 (M1/70.15; Caltag
Laboratories), CD24/heat stable Ag (M1/69; PharMingen, San
Diego, CA), CD25/IL-2R (PC61.5.3, American Type Culture Collection,
Rockville, MD), CD45R/B220 (RA3-6B2; Life Technologies), CD69 (H1.2F3;
PharMingen), TCRß (H57-597; PharMingen), TCR (GL3;
PharMingen), and Gr-1 (RB6-8C5; PharMingen). Donor-derived hemopoietic
cells in bone marrow chimeras were detected using mouse-anti-CD45.2
(104; PharMingen). Streptavidin-phycoerythrin, streptavidin-Red 670
(Life Technologies), and/or streptavidin-allophycocyanin (Caltag
Laboratories) were also used to detect the reactivity of biotinylated
primary Abs. Ab staining and three- or four-color flow cytometric
analyses were conducted using a FACScan and CellQuest software (Becton
Dickinson, San Jose, CA) as previously described (17).
Statistical analysis
Data are presented as the mean ± SD. Statistical significance was assessed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that disorders of the hemopoietic and immune systems and colitis that develops in IL-2-/- mice are initiated by an uncontrolled peripheral activation of T cells by environmental Ags (1, 10), IL-2-/- mice were rederived and maintained in a germfree environment. Histopathologic and microbial analyses of germfree IL-2-/- mice confirmed the absence of contagious pathogens (e.g., Gram-positive and Gram-negative (including Helicobacter sp) bacteria, fungi, and parasites). In addition, serologic tests for Abs against the most common murine pathogens (mouse hepatitis virus, polyoma virus, minute virus of mice, mouse adenovirus, mouse CMV, reovirus type 3, mouse pneumonia virus, Theilers’ virus, Sendai virus, and lymphocytic choriomeningitis virus) were negative.
Up to approximately 8 wk of age, IL-2-/- mice exhibited
no clinical signs of disease, although most were distinguishable from
heterozygote or wild-type littermates by their smaller size and weight.
Between 8 and 12 wk of age, 80% (20 of 25) of IL-2-/-
mice developed anemia (Table I
), the
onset and severity of which varied with age among individual homozygous
mice within the same and different litters. All the germfree
heterozygous and wild-type mice have, to date, been free of any
clinical or histopathologic signs of disease. In addition, a subset (5
of 25) of germfree IL-2-/- mice remained clinically
healthy beyond 6 mo of age. The reason for this heterogeneity in
disease progression is currently not known, but the involvement of any
murine commensal or pathogenic micro-organisms has been excluded.
Visual inspection at autopsy of 8- to 12-wk-old IL-2-/-
mice revealed a moderate to severe splenomegaly and lymphadenopathy,
similar to those seen in SPF IL-2-/- mice. In contrast,
the gastrointestinal tract, with the exception of an enlarged cecum
ordinarily seen in germfree mice, was normal by visual inspection and
histopathology in all germfree IL-2-/- mice analyzed.
Thus, germfree IL-2-/- mice do not develop colitis.
However, they still exhibit signs of immune system and hemopoietic
disorders. To investigate this in further detail, we evaluated
nonlymphoid, lymphoid, and hemopoietic organs for signs of disease.
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The most consistent histopathologic finding of diseased germfree
IL-2-/- mice was a moderate to severe lymphocytic
hyperplasia (Fig. 1) affecting
hemopoietic, lymphoid, and other organs of all
(n = 20) animals analyzed. Among nonlymphoid
tissues, lymphocytic infiltrates were seen in the lungs, liver,
pancreas, and kidneys (Fig. 1, a–d). Chronic
pancreatitis was associated with acinar cell necrosis and atrophy
as well as ductal hyperplasia, although the islets of Langerhans were
unaffected by the inflammatory process. Histochemical analysis of
cytocentrifuge preparations of the cellular infiltrate in the lungs
showed that they comprised atypical lymphocytes, as defined by
their large blast-like appearance, lack of myeloperoxidase, and
presence of tartrate-resistant acid phosphatase (data not shown).
Additionally, occasional foci of cell necrosis in the heart and trachea
were noted in some diseased IL-2-/- mice. The degree of
lymphocytic hyperplasia was most marked in the lymphoid tissues. Lymph
nodes of sick germfree IL-2-/- mice displayed an atypical
follicular hyperplasia, with germinal centers showing poorly defined
borders (data not shown). The spleens of IL-2-/- mice
showed a progressive effacement in architecture due to an expansion of
the white pulp areas and an increase in hemopoietic (erythroid and
myleoid) cells within the red pulp (Fig. 1 and Table II). Compared with the spleen of
IL-2+/+ mice, the red pulp of IL-2-/- mice
was expanded (Fig. 1g). In younger animals (<8 wk of
age), only a mild reactive white pulp hyperplasia was noted.
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Intrahepatic lymphocytes in germfree IL-2-/-mice
Kramer and colleagues (10) have previously described the
accumulation of CD4-CD8+ T cells that
express intermediate levels of the TCRß in the livers of SPF
IL-2-/- mice. Compared with the livers of 5-wk-old
IL-2+/+ littermates, approximately 10-fold more mononuclear
cells were recovered from the livers of germfree IL-2-/-
mice (Fig. 2). Flow cytometric analysis
of these cellular infiltrates showed that the increase in cellularity
in IL-2-/- mice was attributable to increased numbers of
CD4-CD8+ and, to a lesser extent,
CD4+CD8- T cells (Fig. 2). Whereas the number
of CD4+CD8- T cells increased by approximately
13-fold on the average, there was a >30-fold increase in the number of
CD4-CD8+ T cells. Although the
IL-2-/- liver contained proportionately fewer
B220+ (TCR-) B cells than those in littermate
control mice (Fig. 2), when the difference in cellularity of
IL-2-/- and IL-2+/+ liver mononuclear cells
was taken into consideration the numbers of B cells present in germfree
IL-2-/- and IL-2+/+ mice were similar. Also
of note was the >30-fold increase in the number of
B220+TCRßlow/int cells in the livers of
germfree IL-2-/- mice (Fig. 2), a phenotype normally
expressed by activated T cells that accumulate and undergo apoptosis in
the liver (18).
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By 6 wk of age the number of mononuclear cells recovered from the
spleens of IL-2-/- mice increased by 1.5- to 2-fold
compared with that in IL-2+/+ mice. This change was
attributable to increased numbers of both CD4+ and
CD8+ T cells (Fig. 3) and of
mature myeloid (Mac-1high) cells similar to those seen in
SPF IL-2-/- mice (Figs. 2
and 3). The CD4+ T
cells exhibited an activated phenotype, as shown by expression of CD69
and CD25 (Fig. 3A). Unlike the T cells, the
proportion of B cells, as determined by anti-B220 Ab staining, was
decreased in the spleens of 6-wk-old IL-2-/- mice (Fig. 3B). However, when the increase in spleen cellularity
was taken into consideration, there was no significant difference in
the number of B lymphocytes present in the spleens of these animals
compared with that in IL-2+/+ mice (Fig. 3B and
Table II). However, by 8 wk of age the number of B cells in the spleens
of IL-2-/- mice was reduced by approximately 2-fold
compared with that in IL-2+/+ littermates (Table II). Thus,
germfree IL-2-/- mice develop an age-dependent disorder
of the immune system similar to that described in SPF
IL-2-/- mice. We next analyzed the bone marrow of
germfree IL-2-/- mice to assess whether these disorders
affect lymphoid and myeloid development.
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Visual inspection of germfree IL-2-/- mice
revealed brittle bones that upon a more comprehensive analysis
identified striking changes in bone marrow composition that were
evident as early as 4 wk of age (Fig. 4
and Table II). These changes were primarily characterized by a
progressive loss of mature myeloid cells that corresponded to a 20-fold
reduction in mature, Mac-1high and Gr-1high,
polymorphonuclear cells (Table II). The majority of residual cells
expressed low levels of Mac-1/Gr-1 (Fig. 4), characteristic of immature
myeloid cells (19, 20). B cell development was similarly affected in
germfree IL-2-/- mice, as evidenced by the >30-fold
reduction in immature, B220low, and the 10-fold reduction
in more mature, B220high, B cells in the marrow (Fig. 4).
Accompanying the breakdown in B cell development was the infiltration
of large numbers of T lymphocytes that were primarily CD4+
T cells, of which a large proportion was
CD69+CD25+ (Table II). The T cell infiltration
occurred at approximately 6 wk of age, and since the changes in myeloid
cells were seen before this, it is likely that the disruption in bone
marrow hemopoiesis occurs before or coincident with the
lymphoproliferative disorder. The progressive neutropenia seen in the
blood of germfree IL-2-/- mice (Table I) is also
consistent with bone marrow failure. The loss of mature neutrophils was
accompanied by a progressive anemia and the presence of large numbers
of atypical blast-like lymphocytes that were tartrate resistant, acid
phosphatase positive, and myeloperoxidase negative.
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The incidence of colitis in our SPF IL-2-deficient mouse colony
was comparable to that described in other colonies (2). Of the animals
that survived early disease (50%), characterized by splenomegaly,
lymphadenopathy, and anemia, all subsequently developed colitis, with
disease symptoms present between 6 and 10 wk of age. In contrast to the
colitis seen in SPF IL-2-/- mice, none
(n = 25) of the germfree IL-2-/-
mice developed any intestinal pathology. The small and large intestines
of germfree IL-2-/- animals displayed normal
architecture, with no detectable lymphocyte infiltrates, epithelial
cell damage, crypt hyperplasia, or signs of regeneration (Fig. 1, e and f).
Changes in the distribution of iIEL populations
To investigate whether the absence of colitis and gut flora was
accompanied by changes in the intestinal T cell repertoire, we examined
iIELs from both IL-2-/- and IL-2+/+
mice. The iIEL repertoire of the small intestine in
IL-2-/- germfree mice was distinct from that in
littermate control mice. Although the total numbers of iIEL recovered
from IL-2-/- and IL-2+/+ mice were
equivalent, those from IL-2-/- mice contained fewer
CD8-expressing + and ß+ T
cells (Fig. 5B).
Whereas + cells represented the major iIEL population
(63%) in wild-type germfree mice, they comprised only 34% of iIEL in
their IL-2-/- counterparts (Fig. 5A).
Similarly, the number of TCRß+CD8+
iIELs was reduced in germfree IL-2-/- mice, but due to an
increase in CD8ß+ iIEL, the overall number of
TCRß+ iIEL was higher in germfree
IL-2-/- (
1.5 x 106) than that in
IL-2+/+ (1.0 x 106) mice (Fig. 5B). By contrast, the
TCRß+CD4-8- and
TCRß+CD4+ subsets of iIELs did not appear
to be significantly affected by the absence of IL-2. Thus, the absence
of IL-2 results in a decrease in the numbers of CD8+,
particularly + T cells, in the iIEL compartment.
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iIELs to develop in bone marrow radiation
chimeras
To determine whether the decreased numbers of
+ iIELs in germfree IL-2-/- mice
was due to a defect in the ability of these cells to differentiate,
lethally irradiated CD45.1+ (BL/6 congenic) mice were
reconstituted with IL-2-/- (CD45.2+) bone
marrow. The majority of mice (four of five) reconstituted with
IL-2-/- bone marrow survived through 90 days
postreconstitution. At necropsy, tissues were grossly normal, and there
were no signs of colitis in these animals maintained under SPF
conditions (for the duration of the experiment). Analysis of the iIELs
in mice reconstituted with syngeneic (host) bone marrow showed that
these cells were able to fully reconstitute the iIEL compartment (Fig. 6A), such that
ß+ and + iIELs were present in the
same proportions as those seen in unmanipulated wild-type mice (Fig. 5). On the other hand, while IL-2-/- bone marrow was able
to reconstitute the majority (71%) of the iIEL compartment (Fig. 6B), they were unable to fully reconstitute
TCR+ cells. The iIELs present consisted almost
exclusively of ß+ (91%), with very few
+ iIELs (10% or less). Additionally, the
endogenously derived (host) iIELs in IL-2-/- bone
marrow-reconstituted mice were not comprised of ß+ and
+ T cells in the same proportions as those in the
mice reconstituted with syngeneic cells (Fig. 6B).
Since these cells most likely arose from endogenous progenitors that
survived radiation, one possible explanation for this disparity is that
+ iIEL progenitors are more radiosensitive than
ß+ iIEL progenitors. The presence of
TCR+ iIELs in animals reconstituted with syngeneic
bone marrow (Fig. 6A) argues against the possibility
that the absence of these cells in mice reconstituted with
IL-2-/- bone marrow is due to differences in the kinetics
of vs ß iIEL generation and reconstitution. Instead, our
results of iIEL analysis in germfree IL-2-/- mice and
bone marrow chimeras are consistent with a requirement for IL-2 in the
generation of + iIELs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our observations that germfree IL-2-/- mice develop hemopoietic and lymphoid disorders similar to those seen in SPF IL-2-/- mice, with the exception of colitis, demonstrate that environmental Ags are not responsible for the extraintestinal lymphoproliferative disorder and autoimmunity. The ability to distinguish between different mechanisms of disease in IL-2-/- mice is analogous to the distinction between colitis and extraintestinal disease in HLA-B27 transgenic rats (21, 22). In contrast to the inflammatory disorders that affect the intestine, skin, and joints of SPF transgenic rats, the intestine and joints remain histologically normal in germfree animals (22).
The possibility that food Ags could initiate disease in
IL-2-/- mice seems unlikely, since the intestines of
germfree animals remain histologically normal, and abnormal mucosal
immune responses to food Ags would be expected to result in lymphocyte
infiltration and tissue damage, as seen, for example, in celiac’s
disease (23, 24). Instead, our findings are consistent with defects in
intrathymic T cell development and a breakdown in central and/or
peripheral tolerance as the causes of the hemopoietic disorders in
IL-2-/- mice. The accumulation of
B220+TCRlow/int T cells that normally undergo
apoptosis (18) in the livers of germfree IL-2-/- mice and
the defective elimination of superantigen-stimulated peripheral T cells
in IL-2-/- (7) and IL-2R-/- mice (8)
strongly suggest that a breakdown in peripheral tolerance is at least
partially responsible for the disorders seen in IL-2-/-
mice. Additionally, our findings that IL-2 is produced by dying
thymocytes and that activation-induced cell death in the thymi of
IL-2-/- mice is defective (H. Bassiri, P. J. Egan,
E. Samoilova, Y. Chen, and S. R. Carding, unpublished
observations) suggest that a breakdown in thymic tolerance may also be
ultimately responsible for the splenomegaly, lymphadenopathy, and
anemia seen in IL-2-/- mice.
Comparison of the iIEL repertoire in wild-type mice maintained in an
SPF or germfree environment clearly demonstrates the influence of the
gut flora on shaping the iIEL repertoire. The presence of
TCRß+CD8ß+ iIELs appears to be
largely dependent upon bacterial colonization, whereas the
CD8+ subset, which is thought to arise in the gut
mucosa (25), is generated independently of microbial Ags. Among these
cells, the generation or maintenance of TCR+ iIELs
appears to be dependent upon IL-2. The inability of
IL-2-/- bone marrow cells to generate +
iIELs in lethally irradiated hosts is consistent with an intrinsic
defect within hemopoietic or lymphoid progenitor cells. The absence of
TCR+ iIELs in SPF IL-2-/- bone marrow
chimeras demonstrates that the reduced number of these cells in
germfree IL-2-/- mice is not due to autoimmunity, the
absence of gut flora, or differences in food Ags or intestinal
microenvironments that may exist between SPF and germfree mice. The
increased numbers of CDß+ iIELs in germfree
IL-2-/- mice may simply reflect their infiltration into
the epithelia and/or their expansion to fill a niche created by the
failed generation of TCR+ iIELs.
Although we have not been able to identify the defective progenitor
cell(s) in IL-2-/- bone marrow, the presence of
donor-derived TCRß+ iIELs (Fig. 6) and ß T cells
and B cells in the blood6 of
IL-2-/- bone marrow chimeras suggests that the defective
cell resides in a progenitor population rather than in a common
lymphoid progenitor or hemopoietic stem cell population. Furthermore,
the normal number of T cells in extraintestinal tissues of SPF
(26) and germfree (T. Reya, H. Bassiri, and S. R. Carding,
unpublished observations) IL-2-/- mice suggests that the
defect is restricted to a iIEL progenitor population. The
reduced number of TCR+ iIELs in
IL-2Rß-/- mice (14) is consistent with our findings in
IL-2-/- mice and suggests that IL-2/IL-2R signaling may
directly influence iIEL progenitor cell development. Expression
of IL-2Rs by a subset of T lymphoid progenitors within cryptopatches
(27) of the gut is consistent with this interpretation.
The source and utilization of IL-2 within the intestinal mucosa could
be autocrine or paracrine in nature (28, 29), with IL-2 being produced
in response to endogenous and/or food Ags. We cannot, however, exclude
the possibility that IL-2 may influence TCR+ iIELs in
other ways. For example, IL-2 may serve as a survival factor or a
competence factor, enabling iIEL progenitors to acquire
responsiveness to other growth and differentiation factors. These
possibilities are not mutually exclusive, since IL-2 may have multiple
effects during iIEL development, with its effect being
determined by the developmental stage of the IL-2R-bearing cell.
Finally, since the block in iIEL generation is incomplete in
germfree IL-2-/- mice or IL-2-/- bone
marrow chimeras, it is possible that there are compensatory roles for
other factors (30, 31) and/or IL-2-independent pathways of iIEL
development. Regardless of the role of these other factors, our
findings clearly demonstrate the heterogeneity in the requirement for
IL-2 for the development of iIELs and the existence of IL-2-dependent
(TCR+) and independent pathways of iIEL
development.
The absence of colitis in germfree IL-2-/- mice is in
agreement with the previous observations of Sadlack and co-workers (1)
and strongly suggests that enterocolitis is a direct result of an
abnormal immune response in the mucosa to the intestinal bacterial
flora (32, 33, 34). It is possible that TCR iIEL populations, which
are reduced in the absence of IL-2, possess immunoregulatory functions
that contribute to the regulation of mucosal immune responses and to
the maintenance of tolerance to endogenous Ags (35, 36, 37).
In summary, the results of our analysis of germfree IL-2-/- mice show that the anemia and extraintestinal lymphocytic infiltration seen in SPF IL-2-/- mice are most likely the result of uncontrolled responses to endogenous Ags and not to environmental ones. In contrast, the colitis that develops in SPF IL-2-/- mice occurs in response to intestinal bacterial flora. The germfree IL-2-/- mice represent, therefore, a system in which the role of IL-2 in development and regulation of the hemopoietic and immune systems can be assessed in detail in dissociation from the complications of colitis.
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Footnotes |
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2 The first two authors contributed equally to this work.
3 Current address: Department of Microbiology and Immunology, Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94143.
4 Address correspondence and reprint requests to Dr. Simon R. Carding, Department of Microbiology, University of Pennsylvania, 303A Johnson Pavilion, Philadelphia, PA 19104-6076. E-mail address:
5 Abbreviations used in this paper: IL-2-/-, interleukin-2-deficient mice; SPF, specific pathogen free; iIEL, intestinal intraepithelial lymphocytes; int, intermediate level.
6 T. Reya, N. V. Contractor, M. S. Couzens, M. A. Wasik, S. G. Emerson, and S. R. Carding. Abnormal myelocytic cell development in interleukin-2 (IL-2)-deficient mice: evidence for the involvement of IL-2 in myelopoiesis. Submitted for publication.
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chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
T cells. Proc. Natl. Acad. Sci. USA 93:7172. T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
ß-deficient mice fail to develop colitis in the absence of a microbial environment. Am. J. Pathol. 150:91.[Abstract]
T cell receptor positive (TCR+) T cells abrogate oral tolerance, while ß TCR+ T cells provide B cell help. J. Exp. Med. 175:695. T cells. Proc. Natl. Acad. Sci. USA 92:6147. T cell antibody blocks the induction of oral tolerance to ovalbumin in mice. Immunol. Lett. 48:97.[Medline]
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 K. Nakazato, H. Yamada, T. Yajima, Y. Kagimoto, H. Kuwano, and Y. Yoshikai Enforced Expression of Bcl-2 Partially Restores Cell Numbers but Not Functions of TCR{gamma}{delta} Intestinal Intraepithelial T Lymphocytes in IL-15-Deficient Mice J. Immunol., January 15, 2007; 178(2): 757 - 764. [Abstract] [Full Text] [PDF]
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R. Sharma, H. Bagavant, W. N. Jarjour, S.-S. J. Sung, and S.-T. Ju The Role of Fas in the Immune System Biology of IL-2R{alpha} Knockout Mice: Interplay among Regulatory T Cells, Inflammation, Hemopoiesis, and Apoptosis J. Immunol., August 1, 2005; 175(3): 1965 - 1973. [Abstract] [Full Text] [PDF]
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 J. C. I. Singh, S. M. Cruickshank, D. J. Newton, L. Wakenshaw, A. Graham, J. Lan, J. P. A. Lodge, P. J. Felsburg, and S. R. Carding Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G514 - G524. [Abstract] [Full Text] [PDF]
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J. Kang, B. DiBenedetto, K. Narayan, H. Zhao, S. D. Der, and C. A. Chambers STAT5 Is Required for Thymopoiesis in a Development Stage-Specific Manner J. Immunol., August 15, 2004; 173(4): 2307 - 2314. [Abstract] [Full Text] [PDF]
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J.-M. Otte and D. K. Podolsky Functional modulation of enterocytes by gram-positive and gram-negative microorganisms Am J Physiol Gastrointest Liver Physiol, April 1, 2004; 286(4): G613 - G626. [Abstract] [Full Text] [PDF]
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B. H. Nelson IL-2, Regulatory T Cells, and Tolerance J. Immunol., April 1, 2004; 172(7): 3983 - 3988. [Abstract] [Full Text] [PDF]
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J. W. Snow, N. Abraham, M. C. Ma, B. G. Herndier, A. W. Pastuszak, and M. A. Goldsmith Loss of Tolerance and Autoimmunity Affecting Multiple Organs in STAT5A/5B-Deficient Mice J. Immunol., November 15, 2003; 171(10): 5042 - 5050. [Abstract] [Full Text] [PDF]
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T. K. Uren, F.-E. Johansen, O. L. C. Wijburg, F. Koentgen, P. Brandtzaeg, and R. A. Strugnell Role of the Polymeric Ig Receptor in Mucosal B Cell Homeostasis J. Immunol., March 1, 2003; 170(5): 2531 - 2539. [Abstract] [Full Text] [PDF]
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M. Minagawa, H. Watanabe, C. Miyaji, K. Tomiyama, H. Shimura, A. Ito, M. Ito, J. Domen, I. L. Weissman, and K. Kawai Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor {beta}-Chain-Deficient Mice J. Immunol., October 15, 2002; 169(8): 4153 - 4160. [Abstract] [Full Text] [PDF]
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 E. N. Villegas, L. A. Lieberman, S. R. Carding, and C. A. Hunter Susceptibility of Interleukin-2-Deficient Mice to Toxoplasma gondii Is Associated with a Defect in the Production of Gamma Interferon Infect. Immun., September 1, 2002; 70(9): 4757 - 4761. [Abstract] [Full Text] [PDF]
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W. N. D'Souza, K. S. Schluns, D. Masopust, and L. Lefrancois Essential Role for IL-2 in the Regulation of Antiviral Extralymphoid CD8 T Cell Responses J. Immunol., June 1, 2002; 168(11): 5566 - 5572. [Abstract] [Full Text] [PDF]
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L. M. Gaetke, H. S. Oz, W. J. S. de Villiers, G. W. Varilek, and R. C. Frederich The Leptin Defense against Wasting Is Abolished in the IL-2-Deficient Mouse Model of Inflammatory Bowel Disease J. Nutr., May 1, 2002; 132(5): 893 - 896. [Abstract] [Full Text] [PDF]
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M Waidmann, Y Allemand, J Lehmann, S di Genaro, N Bucheler, A Hamann, and I B Autenrieth Microflora reactive IL-10 producing regulatory T cells are present in the colon of IL-2 deficient mice but lack efficacious inhibition of IFN-{gamma} and TNF-{alpha} production Gut, February 1, 2002; 50(2): 170 - 179. [Abstract] [Full Text] [PDF]
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 S. Saitoh, S. Noda, Y. Aiba, A. Takagi, M. Sakamoto, Y. Benno, and Y. Koga Bacteroides ovatus as the Predominant Commensal Intestinal Microbe Causing a Systemic Antibody Response in Inflammatory Bowel Disease Clin. Vaccine Immunol., January 1, 2002; 9(1): 54 - 59. [Abstract] [Full Text] [PDF]
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 M. Yoshida, T. Watanabe, T. Usui, Y. Matsunaga, Y. Shirai, M. Yamori, T. Itoh, S. Habu, T. Chiba, T. Kita, et al. CD4 T cells monospecific to ovalbumin produced by Escherichia coli can induce colitis upon transfer to BALB/c and SCID mice Int. Immunol., December 1, 2001; 13(12): 1561 - 1570. [Abstract] [Full Text] [PDF]
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H. Bassiri and S. R. Carding A Requirement for IL-2/IL-2 Receptor Signaling in Intrathymic Negative Selection J. Immunol., May 15, 2001; 166(10): 5945 - 5954. [Abstract] [Full Text] [PDF]
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 L. V. Hooper and J. I. Gordon Commensal Host-Bacterial Relationships in the Gut Science, May 11, 2001; 292(5519): 1115 - 1118. [Abstract] [Full Text]
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H. C. Rath, M. Schultz, R. Freitag, L. A. Dieleman, F. Li, H.-J. Linde, J. Scholmerich, and R. B. Sartor Different Subsets of Enteric Bacteria Induce and Perpetuate Experimental Colitis in Rats and Mice Infect. Immun., April 1, 2001; 69(4): 2277 - 2285. [Abstract] [Full Text] [PDF]
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J C Hoffmann, K Peters, S Henschke, B Herrmann, K Pfister, J Westermann, and M Zeitz Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after {gamma}{delta} T cell depletion and no effect of {alpha}{beta} T cell depletion Gut, April 1, 2001; 48(4): 489 - 495. [Abstract] [Full Text] [PDF]
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 M. Zareie, P. K. Singh, E. J. Irvine, P. M. Sherman, D. M. McKay, and M. H. Perdue Monocyte/Macrophage Activation by Normal Bacteria and Bacterial Products : Implications for Altered Epithelial Function in Crohn's Disease Am. J. Pathol., March 1, 2001; 158(3): 1101 - 1109. [Abstract] [Full Text] [PDF]
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J. Reddy, P. Chastagner, L. Fiette, X. Liu, and J. Theze IL-2-induced tumor necrosis factor (TNF)-{beta} expression: further analysis in the IL-2 knockout model, and comparison with TNF-{{alpha}}, lymphotoxin-{beta}, TNFR1 and TNFR2 modulation Int. Immunol., February 1, 2001; 13(2): 135 - 147. [Abstract] [Full Text] [PDF]
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L. A. Dieleman, A. Arends, S. L. Tonkonogy, M. S. Goerres, D. W. Craft, W. Grenther, R. K. Sellon, E. Balish, and R. B. Sartor Helicobacter hepaticus Does Not Induce or Potentiate Colitis in Interleukin-10-Deficient Mice Infect. Immun., September 1, 2000; 68(9): 5107 - 5113. [Abstract] [Full Text] [PDF]
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S. Thornton, G. P. Boivin, K. N. Kim, F. D. Finkelman, and R. Hirsch Heterogeneous Effects of IL-2 on Collagen-Induced Arthritis J. Immunol., August 1, 2000; 165(3): 1557 - 1563. [Abstract] [Full Text] [PDF]
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 S. Rao and A. S. Verkman Analysis of organ physiology in transgenic mice Am J Physiol Cell Physiol, July 1, 2000; 279(1): C1 - C18. [Abstract] [Full Text] [PDF]
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 D. A. Mark, C. E. Donovan, G. T. De Sanctis, H. Z. He, M. Cernadas, L. Kobzik, D. L. Perkins, A. Sharpe, and P. W. Finn B7-1 (CD80) and B7-2 (CD86) Have Complementary Roles in Mediating Allergic Pulmonary Inflammation and Airway Hyperresponsiveness Am. J. Respir. Cell Mol. Biol., March 1, 2000; 22(3): 265 - 271. [Abstract] [Full Text]
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Y.-G. Lai, V. Gelfanov, V. Gelfanova, L. Kulik, C.-L. Chu, S.-W. Jeng, and N.-S. Liao IL-15 Promotes Survival But Not Effector Function Differentiation of CD8+ TCR{alpha}{beta}+ Intestinal Intraepithelial Lymphocytes J. Immunol., December 1, 1999; 163(11): 5843 - 5850. [Abstract] [Full Text] [PDF]
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B. O. Porter and T. R. Malek IL-2R{beta}/IL-7R{alpha} Doubly Deficient Mice Recapitulate the Thymic and Intraepithelial Lymphocyte (IEL) Developmental Defects of {gamma}c-/- Mice: Roles for Both IL-2 and IL-15 in CD8{alpha}{alpha} IEL Development J. Immunol., December 1, 1999; 163(11): 5906 - 5912. [Abstract] [Full Text] [PDF]
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S. Marguerat, H. R. MacDonald, J.-P. Kraehenbuhl, and J. P. M. van Meerwijk Protection from Radiation-Induced Colitis Requires MHC Class II Antigen Expression by Cells of Hemopoietic Origin J. Immunol., October 1, 1999; 163(7): 4033 - 4040. [Abstract] [Full Text] [PDF]
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H. K. Wong, G. M. Kammer, G. Dennis, and G. C. Tsokos Abnormal NF-{kappa}B Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus Is Associated with Decreased p65-RelA Protein Expression J. Immunol., August 1, 1999; 163(3): 1682 - 1689. [Abstract] [Full Text] [PDF]
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Y. Umesaki, H. Setoyama, S. Matsumoto, A. Imaoka, and K. Itoh Differential Roles of Segmented Filamentous Bacteria and Clostridia in Development of the Intestinal Immune System Infect. Immun., July 1, 1999; 67(7): 3504 - 3511. [Abstract] [Full Text] [PDF]
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H. C. Rath, K. H. Wilson, and R. B. Sartor Differential Induction of Colitis and Gastritis in HLA-B27 Transgenic Rats Selectively Colonized with Bacteroides vulgatus or Escherichia coli Infect. Immun., June 1, 1999; 67(6): 2969 - 2974. [Abstract] [Full Text] [PDF]
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M. Schultz, S. L. Tonkonogy, R. K. Sellon, C. Veltkamp, V. L. Godfrey, J. Kwon, W. B. Grenther, E. Balish, I. Horak, and R. B. Sartor IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation Am J Physiol Gastrointest Liver Physiol, June 1, 1999; 276(6): G1461 - G1472. [Abstract] [Full Text] [PDF]
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C.-L. Chu, S.-S. Chen, T.-S. Wu, S.-C. Kuo, and N.-S. Liao Differential Effects of IL-2 and IL-15 on the Death and Survival of Activated TCR{gamma}{delta}+ Intestinal Intraepithelial Lymphocytes J. Immunol., February 15, 1999; 162(4): 1896 - 1903. [Abstract] [Full Text] [PDF]
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R. K. Sellon, S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, E. Balish, D. M. Rennick, and R. B. Sartor Resident Enteric Bacteria Are Necessary for Development of Spontaneous Colitis and Immune System Activation in Interleukin-10-Deficient Mice Infect. Immun., November 1, 1998; 66(11): 5224 - 5231. [Abstract] [Full Text] [PDF]
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 T. Reya, N. V. Contractor, M. S. Couzens, M. A. Wasik, S. G. Emerson, and S. R. Carding Abnormal Myelocytic Cell Development in Interleukin-2 (IL-2)-Deficient Mice: Evidence for the Involvement of IL-2 in Myelopoiesis Blood, April 15, 1998; 91(8): 2935 - 2947. [Abstract] [Full Text] [PDF]
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A. Fischer and B. Malissen Natural and Engineered Disorders of Lymphocyte Development Science, April 10, 1998; 280(5361): 237 - 243. [Abstract] [Full Text]
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