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B-Inducing Kinase1
*
First Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan; and
Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-inducing kinase (NIK) gene, manifest a unique phenotype;
they lack lymph nodes and Peyer’s patches, have a disturbed spleen
architecture, and exhibit defects in both Ab and cellular immune
responses. Although a stromal defect caused by impaired lymphotoxin-ß
receptor signaling accounts for their abnormal lymphoid organogenesis,
the exact mechanisms underlying the development of immunodeficiency in
aly mice are poorly understood. We therefore
investigated the contribution of hemopoietic cells with the
aly NIK mutation to the development of immunodeficiency.
Transfer of aly/aly bone marrow cells
into aly/+ mice resulted in poorly developed B cell
follicles and lack of support for the development of germinal centers
and isotype switching, indicating that the hemopoietic cells of
aly mice contain an autonomous defect. However,
follicular dendritic cell clusters were maintained in the spleens of
these bone marrow chimeras, suggesting that the lack of follicular
dendritic cell clusters in aly mice is probably due to
the stromal defect. The aly mice lacked marginal zone B
cells in their spleens, and aly/aly B
cells showed an impaired proliferative response after in vitro
stimulation. IL-2 production by activated T cells was also impaired. By
contrast, the dendritic cells of aly mice exhibited
grossly normal development and function. Supporting the concept of an
autonomous cell defect, Rel protein expression was altered in
aly/aly spleens. Thus, the
aly NIK mutation affects hemopoietic cell function in an
intrinsic fashion and, together with the stromal defect, may contribute
to the development of immunodeficiency in aly
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, a positional cloning study has identified the
NF-B-inducing kinase (NIK) gene to be responsible for the
aly mutation (4). NIK was originally identified
as a gene that participates in the NF-B-inducing signaling cascade
induced by TNF, CD95, and IL-1 (5). Subsequently, NIK was
demonstrated to phosphorylate both IB kinase-
and IB
kinase-ß, which may sequentially activate the downstream targets
necessary for NF-B activation (6, 7, 8, 9, 10, 11). We have
previously shown that signaling via the lymphotoxin-ß receptor
(LTßR) is impaired in aly mice; no up-regulation of VCAM-1
occurred in aly mouse embryonic fibroblasts after
stimulation with an agonistic anti-LTßR mAb (12),
suggesting that NIK is also involved in LTßR signaling. Because
LTßR signaling appears to be essential for the development of
lymphoid organs (1, 13), its impairment by the
aly NIK mutation accounts for the abnormal development of
the lymphoid organs in this strain (4, 12). We have also
investigated the mechanisms underlying defective lymphoid organogenesis
in aly mice by generating aggregation chimeras. These
studies demonstrated that the abnormal development of the lymphoid
organs in aly mice is probably due to defective development
of the precursor stromal cells of the LN and PP (12).
Because LTßR is exclusively expressed by nonlymphoid cells
(14), the defective LTßR signaling present in
aly mice implies that the absence of lymphoid organogenesis
in this strain is caused by a defect in non-bone marrow (BM)-derived
cells (12).
It has been demonstrated that the ability to induce an efficient anti-viral response is compromised in aly mice, probably due to the abnormal development of the secondary lymphoid organs as well as to the disorganized spleen architecture (15). However, experiments with BM transfer have also suggested that the BM-derived cells of aly mice function abnormally; after aly mice received BM from wild-type mice, their serum Ig levels increased into the normal range (2). Because aly mice manifest a compound phenotype, NIK mutation, and lack of LN, PP, and FDC clusters in the spleen (2, 3, 4), it has been difficult to determine whether the defect in their immune function results from abnormal BM-derived cells such as B and T cells. Furthermore, it remains unclear how BM-derived cells with a NIK mutation may contribute to the development of immunodeficiency. In the present study we addressed this question using a BM transfer system and clearly demonstrated that BM-derived cells from aly mice have an autonomous cell defect; transfer of aly/aly BM cells into aly/+ mice resulted in poorly developed B cell follicles together with lack of support for the development of GC and isotype switching even in the presence of both peripheral lymphoid organs and FDC clusters in the spleen. Supporting the concept of an autonomous defect in hemopoietic cells, the function of both B cells and T cells was impaired (as assessed by the in vitro culture system), and Rel protein expression by the spleen cells was altered in aly mice. Thus, NIK plays an important role in the development and/or function of BM-derived cells as well as in the development of non-BM-derived cells; the impaired Ab response seen in aly mice is associated not only with a lack of FDC clusters in the spleen (caused by a defect in non-BM-derived cells), but also with an intrinsic defect in lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aly/+ mice, aly/aly mice (2), BALB/c, and C57BL/6J mice were purchased from CLEA Japan (Osaka, Japan). The mice were maintained under pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experimentation of Tokushima University School of Medicine. The experiments were initiated when the mice were 8–12 wk of age.
BM transfer
BM transfer was performed as described previously (16). In brief, BM cells were harvested by flushing the femurs of donor mice with RPMI 1640 medium (Life Technologies/BRL, Grand Island, NY) supplemented with 10% heat-inactivated FBS (Life Technologies/BRL), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, hereafter referred to as R10. The cells were washed once and suspended in R10 medium containing anti-CD90 (Thy1.2) mAb (clone 5a-8; Cedarlane Laboratories, Ontario, Canada) plus low toxicity rabbit C (Cedarlane Laboratories). After incubation at 37°C for 45 min, the cells were washed twice and adjusted to 3 x 107 viable cells/ml in R10. Each recipient mouse was lethally irradiated (10 Gy) and treated with 0.5 ml of donor BM cells i.v. on the same day. The recipient mice were used in the analyses 6–10 wk after BM transfer.
Measurement of anti-SRBC response
Mice were immunized i.p. with 100 µl of a 10% SRBC suspension in PBS. Ten days later sera were collected, and specific Abs were measured by ELISA as described previously (17). In brief, microtitration plates (ICN Biomedicals, Aurora, OH) were coated with SRBC (150 µl at 5 x 107 cells/ml) suspended in 0.25% glutaraldehyde in PBS. The diluted mouse sera were added, and bound Abs were detected with alkaline phosphatase-conjugated goat anti-mouse isotype-specific antisera (Southern Biotechnology Associates, Birmingham, AL). The mean OD measured in triplicate wells was compared with a standard curve constructed from titrated serum values to calculate the response in relative units using linear regression analysis. The results are expressed as the mean ± SEM.
Immunohistochemistry
Ten days after i.p. injection of 100 µl of a 10% SRBC suspension in PBS, the mouse spleens were harvested, and frozen sections were stained with anti-CD45R/B220 mAb, anti-CD90 (Thy1.2) mAb, anti-CD35 (C receptor 1; CR1) mAb (clone 8C12; PharMingen, San Diego, CA), and peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA) as previously described (16, 18).
Flow cytometric analysis
Spleen cell suspensions were prepared by teasing the tissues apart between two frosted microscope slides. The suspensions were depleted of RBC by osmotic lysis, and the cells were stained with the following mAbs: anti-CD45R/B220, anti-IgM, anti-IgD and anti-CD35 (PharMingen). The cells were analyzed using an EPICS Elite (Coulter, Hialeah, FL) or a FACScalibur (Becton Dickinson, San Jose, CA) flow cytometer with CellQuest software, as described previously (12).
Proliferative response of B cells
The spleen cell suspensions were depleted of RBC by osmotic
lysis, and their B cells were purified with MACS CD45R (B220)
microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to
the manufacturer’s instructions. The resulting preparations contained
95 and 85% CD45R/B220+ cells from
aly/+ mice and aly/aly mice,
respectively. No contaminating T cells from either aly/+ or
aly/aly mice were detectable. The purified B
cells were cultured in 0.2 ml of R10 medium containing 50 µM 2-ME at
a density of 1 x 105 or 1.5 x
105 cells/well in flat-bottom 96-well plates. The
cells were stimulated with 10 µg/ml LPS (Sigma, St. Louis, MO), 10
µg/ml affinity-purified goat anti-IgM Ab (ICN Pharmaceuticals),
and 10 µg/ml rat agonistic anti-CD40 mAb (clone 3/23; Serotec,
Oxford, U.K.). For the last 8 h of the 48-h culture period the
cultures were pulsed with 0.5 µCi/well
[3H]thymidine (NEN, Boston, MA). They were then
harvested onto glass-fiber filters, and the radioactivity was measured
in a beta counter (LS 6500, Beckman, Fullerton, CA).
In vitro Ig production
Purified B cells (1 x 105 cells/well) were stimulated with either 10 µg/ml LPS or 10 µg/ml agonistic anti-CD40 mAb plus 10 ng/ml rIL-4 (PeproTech, London, U.K.) for 6 days. The concentrations of Ig in the culture supernatants were then measured by a sandwich ELISA using a clonotyping system with HRP (Southern Biotechnology Associates).
Proliferative response of T cells
Mononuclear cells from the spleen were first purified using
Histopaque-1119 (Sigma), then were incubated in a plastic dish for 45
min in a humidified incubator. Any nonadherent cells were removed, then
the T cells were isolated using the Cellect Mouse T Cell Kit (Cytovax
Biotechnologies, Alberta, Canada) according to the manufacturer’s
instructions. The resulting preparations contained 85% CD3-positive
cells from both aly/+ and aly/aly
mice, and no contaminating B cells were detectable. The purified T
cells (2 x 105 cells/well) were stimulated
with immobilized anti-CD3 mAb (clone 145-2C11; PharMingen) or
anti-CD3 mAb plus anti-CD28 mAb (clone 37.51; Serotec). For the
last 8 h of the 72-h culture period, the cells were pulsed with
0.5 µCi/well [3H]thymidine, and
3H incorporation was determined as described
above. IL-2 production by the culture supernatants was
determined using an ELISA kit (Amersham, Aylesbury, U.K.) after 72
h of culture.
Mixed lymphocyte reactions
T cell-depleted spleen cells from BALB/c mice were prepared with Dynabeads (mouse pan T, Thy1.2; Dynal, Oslo, Norway) and irradiated at 20 Gy. These cells (5 x 105) were then mixed with purified T cells (2 x 105) and cultured in flat-bottom 96-well plates. For the last 8 h of the 72-h culture period the cultures were pulsed with 0.5 µCi/well [3H]thymidine. In the same set of experiments the supernatants were harvested after 72 h of culture for measurement of IL-2 production.
Isolation of dendritic cell (DC)-enriched populations from the spleen
DC-enriched populations were isolated from the spleen as described previously (19). In brief, a collagenase-digested splenocyte suspension was overlaid onto dense BSA solution (1.080 g/ml) and centrifuged for 15 min at 9500 x g. The low density splenocytes were recovered and subjected to plastic adherence for 90 min. After removing the nonadherent cells, the adherent cells were incubated overnight, after which any cells that became detached were recovered as DC-enriched populations. These populations contained >60% CD11c+ cells; the rest consisted mainly of macrophages. These cells (1 x 104) were used as the allogeneic stimulator during MLR, as described above.
Allogeneic skin graft rejection
Approximately 1 x 1-cm pieces of tail skin were obtained from aly/+ or aly/aly mice and were grafted onto the lateral thoracic region of anesthetized BALB/c mice using a single piece of skin per recipient mouse. The bandages were removed 6 days later, and the grafts were observed daily. Rejection was recorded as the first day on which scab formation became visible, which was usually 2–3 days before complete rejection of the graft.
Western blot analysis
Whole cell lysates were prepared from the RBC-lysed spleen
cells, using a lysis buffer containing 1% Nonidet P-40 (Sigma), and
their proteins were separated by SDS-PAGE, blotted onto Hybond-P
transfer membranes (Amersham) using a semidry blotter (Bio-Rad,
Hercules, CA), and analyzed using an enhanced chemiluminescence Western
blotting detection system (Amersham). The Abs used were rabbit
anti-peptide Abs directed against p50 (catalogue no. sc-114), p52
(catalogue no. sc-298), RelA (catalogue no. sc-109), RelB (catalogue
no. sc-226), c-Rel (catalogue no. sc-71), IB (catalogue no.
sc-371), and IBß (catalogue no. sc-945), all purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aly mice have been demonstrated to have an impaired Ab response when immunized with T cell-dependent Ags; specific Ab titers are very low, and isotype switching is defective (3). It remains unclear, however, whether the defect is intrinsic to the hemopoietic cells, caused by the abnormal development of the secondary lymphoid organs, or both. Using reciprocal BM transfers between aly/+ and aly/aly mice, we investigated the mechanisms underlying the defective Ab response in aly mice by analysis of specific Ab responses together with histological evaluation of the spleen after BM transfer.
The aly/+ spleen exhibited well-organized T cell/B cell
segregation (Fig. 1
A) and FDC
clusters (Fig. 1E), and GC formation was observed after
immunization with SRBC without adjuvant (Fig. 1I). By
contrast, the aly/aly spleen showed poorly
developed B cell follicles (Fig. 1B), and no FDC clusters or
GC formation were observed (Fig. 1, F and
J).
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). After immunization, PNA-positive cells
were observed at ectopic sites in these chimeric mice (Fig. 1K); however, in contrast to aly/+ mice, these
PNA-positive cells were not confined to areas composed only of B cells,
but were also located in areas containing T cells (compare K
with I in Fig. 1). There were no FDC clusters (identified by
anti-CD35 mAb) after transfer of aly/+ BM (Fig. 1G), suggesting that the lack of FDC clusters in
aly mice is due to a defect in non-BM-derived cellular
components. When specific Ab responses were assessed in these chimeras,
we found that the IgM class anti-SRBC response was even higher than
that in aly/+ mice (Fig. 2).
The IgG1 class anti-SRBC response, however, remained undetectable,
as in aly/aly mice. Thus, aly/+ cells cannot
undergo normal class switching in the aly/aly
mouse environment (i.e., in the absence of FDC clusters), although they
can become PNA positive.
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D). Although FDC clusters (probably of
aly/+ mouse origin) were observed in these chimeras (Fig. 1H), as in aly/aly mice, no
PNA-positive cells appeared after immunization (Fig. 1L). As
also seen in aly/aly mice, both IgM class and
IgG1 class anti-SRBC responses were very low in this group of BM
chimeras (Fig. 2). These results clearly demonstrate that
aly/aly BM cells contain an autonomous defect,
which was exemplified by their inability to populate aly/+
mouse spleen and by their defective Ab responses even in the
aly/+ mouse environment (i.e., in the presence of FDC
clusters). Absence of marginal zone B cells and altered CR expression in aly mice
The BM transfer experiment described above strongly suggested that
the aly NIK mutation affects B cell development and/or
function. We therefore characterized B cells from aly mice,
first by examining their cell surface markers using flow cytometry. As
reported previously, there were only 30% as many
B220+ cells in the aly/aly
spleen as in the aly/+ spleen (2, 3) (Fig. 3A, top). Despite
this reduction in numbers, the B220+ cells from
aly/aly mice expressed similar levels of B cell
markers (such as CD19, CD40, I-Ab, and CD80) as
those from aly/+ mice (data not shown). The CR expression
profile, however, exhibited major differences in
aly/aly and aly/+ spleen cells. In
aly/+ spleen, most of the B220+ cells
expressed CD35 (Fig. 3A, top), and both
CD35low and CD35high
populations could be identified (Fig. 3B). It has previously
been shown that B cells lying within follicles are
CD21/35low, whereas those from the marginal zone
are CD21/35high (20). Although most
of the B220+ cells from
aly/aly mice were also CD35 positive, there were
no CD35high populations (Fig. 3, A,
top, and B), which is consistent with the lack of
a marginal zone structure in aly mice (15, 21).
A lack of cells characteristic of marginal zone B cells in
aly/aly mice was also demonstrated by the absence
of CD35highIgDlow
populations (Fig. 3A, middle). Likewise, there
were no CD35highIgMhigh
populations in spleens from aly/aly mice (data
not shown). Interestingly, the amount of CD35 expressed on
B220+ cells from aly/aly
mice was even lower than that in CD35low
populations among aly/+ mice (Fig. 3B). This
reduced CD35 expression by aly/aly splenic B
cells was not, however, associated with alterations in other B cell
differentiation markers; there were similar percentages of
IgM+IgD+ cells among the
B220+ cells in both aly/+ and
aly/aly mice (data not shown). By contrast, the
percentage of B220+IgM+
cells in aly/aly BM was significantly reduced
compared with that in aly/+ BM (Fig. 3A,
bottom), suggesting that the NIK mutation affects B cell
maturation in the BM rather than at a later stage in the periphery.
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B cell function in aly mice was assessed using
B220+ cells isolated from the spleen. B cells
from aly/aly mice showed <50% of the
proliferative responses seen in aly/+ B cells after
stimulation with LPS, anti-IgM, and anti-CD40 (Fig. 4). Because of the slightly lower purity
of the B cell preparation from aly/aly spleen, we
repeated the experiment using 1.5 times more
aly/aly B cells in the culture. Reduced
[3H]thymidine incorporation was still observed
after stimulation with LPS and anti-CD40. Thus, the NIK mutation
affects not only B cell development but also B cell function, further
supporting the concept of an autonomous defect in the hemopoietic cells
of aly mice.
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). Despite the impaired proliferative
response of these aly/aly B cells, there was
comparable IgM, IgG2b, and IgG3 production after LPS stimulation and
IgG1 production after anti-CD40 plus IL-4 stimulation between
aly/+ and aly/aly mice. Thus, the
effect of the NIK mutation on B cells depends on the specific function
tested.
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Although aly mice have a disturbed thymic structure,
flow cytometric analysis using CD4 and CD8 mAbs did not show any
significant alterations in the thymocyte population (2).
In the spleen the CD4/CD8 ratio showed only a small reduction in
aly mice (data not shown). A previous report of the failure
of allogeneic skin graft rejection in aly mice
(2), however, strongly suggests that T cell function may
be compromised by the NIK mutation. We therefore examined the function
of purified T cells from aly/aly spleen using an
in vitro culture system. Although the proliferative response of
aly/aly T cells after stimulation with
immobilized anti-CD3 showed only a subtle change, IL-2 production
was dramatically reduced compared with that in aly/+ T cells
(Table II). Because it has been suggested
that NIK plays an important role in NF-B activation through the
CD3/CD28 stimulation pathway (22), we also investigated
whether the impairment in IL-2 production by anti-CD3-activated
aly/aly T cells could be restored by coligation
with CD28. Although still reduced compared with that by
aly/+ T cells, IL-2 production by
aly/aly T cells was significantly increased after
CD28 coligation.
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B), while the proliferative
response was similar in aly/+ and
aly/aly mice (Fig. 5A). TNF and
IFN-
release into the culture supernatants during allogeneic MLR
were comparable in aly/+ and aly/aly
mice (data not shown). Thus, aly/aly T cells
showed impaired IL-2 production upon stimulation. By contrast, these
cells could proliferate normally in response to exogenous IL-2 (data
not shown).
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We next assessed whether the NIK mutation affects the development
and/or function of DCs. Consistent with the results of previous studies
(15), immunohistochemical analyses of the spleen using DC
markers showed no obvious defect in DC development in aly
mice; NLDC145-, MIDC8-, and CD11c-positive cells were present to a
similar extent in aly/+ and aly/aly
mice despite the disorganized spleen architecture in aly
mice (data not shown). We then prepared DC-enriched fractions from the
spleen using gradient separation in combination with adherence to a
plastic dish. In both aly/+ and
aly/aly mice, these fractions contained similar
percentages of CD11c+ cells. These cells were
tested for the ability to stimulate allogeneic T cells from BALB/c
mice. DC-enriched fractions from aly/+ and
aly/aly mice stimulated T cell proliferation to
similar levels, suggesting that DC function is retained in
aly mice (Fig. 6). After
stimulation with anti-CD40 mAb, the DC-enriched fractions from
aly/aly mice also formed cellular aggregations in
a similar manner as those from aly/+ mice (data not
shown).
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). During the same set of experiments,
BALB/c skin grafted onto BALB/c mice produced no signs of rejection for
>2 mo (data not shown).
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Because NIK has been demonstrated to play a critical role in
NF-B activation, and the expression of Rel proteins is regulated by
NF-B complexes (24, 25, 26, 27), we assessed the expression of
various Rel proteins in aly/aly spleen cells by
Western blotting. p52 expression was significantly reduced in
aly/aly mice, although the expression of p100, a
precursor form of p52, was comparable in aly/+ and
aly/aly mice (Fig. 7A). Spleen cells from
aly/aly mice that had received aly/+
BM contained similar levels of p52 as those from untreated
aly/+ mice, suggesting that reduced expression of p52 is
intrinsic to aly/aly BM-derived cells.
Conversely, spleen cells from aly/+ mice that had received
aly/aly BM contained little p52, but retained the
ability to produce p100 protein, as observed in untreated
aly/aly mice. A significant reduction in c-Rel
expression and moderate reductions in p50, RelA, and RelB were also
observed in aly mice (Fig. 7B). IB and
IBß expression were comparable in aly/+ and
aly/aly mice. The aly/+ spleen
contained levels of Rel and IB proteins indistinguishable from those
in C57BL/6 mice (Fig. 7B). Taken together, the altered
expression patterns of the various Rel proteins further support the
concept of an autonomous defect in the hemopoietic cells of
aly mice, which may account for the alterations in
lymphocyte function in this strain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BM transfer experiments employed in the present study revealed interesting aspects of the relationship between lymphoid organ development and Ab responses. After receiving aly/+ BM, aly/aly mice showed a greater IgM response than aly/+ mice. This greater IgM response, however, was not accompanied by IgG class switching when PNA-positive cells were present in the spleen, but there were no detectable FDC clusters. These results suggest that FDC clusters play a critical role in the efficient switching of Ab production. Conversely, aly/+ mice that received aly/aly BM cells exhibited poorly developed B cell follicles together with lack of support for the development of GC and isotype switching. This defective Ab response occurred despite the presence of both FDC clusters in the spleen (probably of aly/+ recipient mouse origin) and an LN structure (populated by cells from aly/aly mice), suggesting that the impaired Ab response in aly mice does not result only from the absence of FDC clusters in the spleen and/or from the lack of an LN structure. Thus, the present results underscore the contribution of the autonomous defect in BM-derived cells with the NIK mutation to the development of immunodeficiency in aly mice.
Because marginal zone B cells are uniquely positioned near the marginal
sinus, where the extensive blood flow facilitates their exposure to
blood-borne Ags, both the marginal zone structure and marginal zone B
cells are considered important for effective Ab responses
(20). It has been demonstrated that the aly
mouse spleen lacks a marginal zone structure; no mucosal addressin cell
adhesion molecule-1-positive cells were found at the site corresponding
to the marginal zone, and the sinus structure was ambiguous
(21). This lack of a marginal zone structure was
associated with the absence of MOMA-1 (anti-metallophilic
macrophage)-positive macrophages in this area (15, 21). In
the present study we have also demonstrated that aly mouse
spleen lacks B cells that possess the characteristics of marginal zone
B cells (i.e., high expression of both CR and IgM, but low IgD
expression). The relationship between the marginal zone structure and
the generation of marginal zone B cells merits attention.
Lymphotoxin--deficient mice also lack a marginal zone, and no
mucosal addressin cell adhesion molecule-1-positive cells or
MOMA-1-positive macrophages exist at the site corresponding to the
marginal zone (16). Like those of aly mice, the
spleens of lymphotoxin--deficient mice produce no B cells
characteristic of marginal zone B cells (our unpublished observations).
Thus, the generation of marginal zone B cells is closely related to the
presence of a normal marginal sinus structure, the formation of which
is dependent on signals transmitted through the LTßR and expressed by
the stromal element. A requirement for NIK in the critical dialogue
between BM-derived cells and their microenvironment therefore seems
likely.
Although aly mice lack B cells with high CD35 expression, as
described above, their B cells do express reduced levels of CD35 (Fig. 3B). Because CR have been implicated as important regulators
of B cell response (28), it is possible that this reduced
expression of CR on B cells may contribute to the impaired Ab response
in aly mice. However, we speculate that reduced CR
expression does not in itself play a major role in the impairment of Ab
responses in this strain for the following reason. Previous research
has shown that B cells from mice heterozygous for inactivation of the
CR1/2 gene (CR1/2+/-) exhibit similar reduced CR
expression profiles as those seen in aly mice; there were no
CD35high populations, and the level of CD35
expressed was lower than that seen in a CD35low
population from wild-type mice because of the gene dosage effect
(29). However, despite the reduced CR expression,
CR1/2+/- mice generally showed normal IgM and
IgG responses after immunization with SRBC (H. Molina, personal
communication). Therefore, other factors, such as lack of a marginal
zone and/or impaired B cell signaling (see below), may be more relevant
to the defective Ab response in aly mice. The exact
mechanism by which NIK affects CR expression and its pathophysiological
relevance require further investigation.
Using an in vitro culture system we have demonstrated that B cells from
aly mice have impaired proliferative responses upon
stimulation with LPS, anti-IgM, and anti-CD40. Because marginal
zone B cells have been shown to proliferate better than follicular B
cells when stimulated with LPS or CD40 ligation (30), the
impaired proliferative response upon LPS or anti-CD40 stimulation
in aly mice could be due merely to the lack of marginal zone
B cells in the B cell preparation. However, B cells from
lymphotoxin--deficient mice, which also lack marginal zone B cells,
as discussed above, show no impairment of proliferative responses after
treatment with these stimulants (our unpublished observations). We
therefore speculate that the impaired B cell proliferative response in
aly mice is due to an intrinsic defect in the B cells
per se.
We have also demonstrated that T cell function is compromised in
aly mice, and that IL-2 production by activated T cells is
dramatically reduced. Because aly/aly T cells
were able to proliferate after anti-CD3 stimulation or during
allogeneic MLR, this impaired IL-2 production is not due to complete
disruption of TCR/CD3 signaling in aly mice. One line of
evidence suggesting that IL-2 production is controlled by NF-B
activation is that T cells from c-Rel-deficient mice show impaired IL-2
production in response to anti-CD3 stimulation (31).
Because the aly/aly spleen contains reduced
levels of c-Rel protein (Fig. 7B), it may be reasonable to
speculate that the impaired IL-2 production in aly mice is
associated with reduced c-Rel expression, which, in turn, is caused by
the NIK mutation.
It has been demonstrated that CD28 cooperates with TCR/CD3 in the
activation of AP-1 and NF-B, which have cognate binding sites in the
CD28 response element of the IL-2 gene promoter (32, 33, 34).
It has also been demonstrated that NIK plays an important role in
NF-B activation through CD3/CD28 stimulation (22).
Inconsistent with the latter finding, we have observed significant
restoration of IL-2 production after CD28 coligation in aly
mice. This discrepancy could be due simply to a point mutation (as
opposed to a null mutation) of NIK in aly mice, although the
full restoration of aly mouse phenotypes by transgenic
expression of wild-type NIK does not support this possibility
(4). Alternatively, NIK may participate in NF-B
activation or target gene activation by other undefined mechanisms.
Further studies are required to reach a full understanding of the roles
played by NIK in gene regulation (35).
In contrast to the impairment of lymphocyte function in aly mice, tests of DC function have to date revealed no obvious defect. The aly/aly DCs were able to stimulate allogeneic T cells to a similar degree as those from aly/+ mice during MLR. Retainment of the function of DC in aly mice in vivo was also suggested by skin-grafting experiments; skin APCs from aly/aly mice were equally able to induce host T cell activation in vivo as those from aly/+ mice. These results suggest that DC function is retained in aly mice, although other functional aspects of DCs in which NIK might be involved have yet to be examined.
Consistent with the concept of an intrinsic defect in hemopoietic cells, we found that aly mice have altered expression of many Rel proteins in the spleen; the most prominent reductions were observed for p52 and c-Rel. In this respect, it is interesting to note that aly mice and p52-deficient mice share some common phenotypic features; both strains lack FDC clusters and GC formation in the spleen, and both exhibit an impaired Ab switching response to a T cell-dependent Ag (36, 37). Furthermore, impaired proliferative responses of the B cells in the context of apparently normal Ig production were observed in both strains upon in vitro stimulation (our present study and Ref. 37). Although these similarities raise the possibility that the phenotypic characteristics of aly mice could be associated with reduced p52 production, it is important to point out that there is a clear difference between the two strains. Adoptively transferred BM cells from p52-deficient mice have been shown to support GC formation as well as a switched Ab response in recombinase-activating gene-1-deficient mice (36). Based on these results, it was suggested that the primary defects underlying these biologic processes reside not within the mature p52-deficient B or T cell lineages, but, rather, within accessory cells (36). In contrast to the findings in p52-deficient mice, adoptively transferred BM cells from aly/aly mice supported neither GC formation nor a switched Ab response in aly/+ mice. Furthermore, intrinsic defects in both B and T cells were observed in aly mice using an in vitro culture system. Thus, NIK may affect the function of a broader spectrum of cell types than p52. Although less dramatic compared with the reduction in p52, reduced levels of p50 were observed in aly mice, although production of p105 (a precursor form of p50) was retained. It is therefore intriguing to speculate that NIK regulates the processes involved in the generation of p50 and p52 from their precursor proteins and/or the turnover of p50 and p52 proteins.
Finally, it is important to emphasize that the exact mechanisms underlying the lack of allogeneic skin graft rejection in aly mice remain unsolved. Because mice deficient in IL-2 can reject pancreatic islet allografts and generate an effective CTL response to allogeneic tumor cells (38), it seems unlikely that impaired IL-2 production fully explains the lack of allogeneic skin graft rejection in aly mice. Although some responses to signals transmitted through CD40 are attenuated, as demonstrated in this study, up-regulation of CD86 on CD40-activated B cells has been observed in aly mice (our unpublished observations). In addition, the CD40 ligand is expressed normally on activated T cells in aly mice (our unpublished observations), and CD28 signaling is retained, as described above. Thus, two principal pathways that control the induction of allograft rejection (i.e., CD40 ligand-CD40 and B7-CD28) (39) do not appear to be disrupted by the aly NIK mutation. Further studies of the mechanisms by which this NIK mutation affects allograft rejection processes may eventually allow better strategies to be developed for the control of organ transplantation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mitsuru Matsumoto, Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
3 Abbreviations used in this paper: LN, lymph node; PP, Peyer’s patch; LTßR, lymphotoxin-ß receptor; NIK, NF-B-inducing kinase; GC, germinal center; FDC, follicular dendritic cell; BM, bone marrow; PNA, peanut agglutinin; DC, dendritic cell; LC, Langerhans cell.
Received for publication January 6, 2000. Accepted for publication April 27, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or
aly/aly mice (1 x 105
cells/well, 
; 1.5 x 105 cells/well, 
) were
cultured with or without the stimulants indicated. For the last 8
h of the 48-h culture period, the cultures were pulsed with
[3H]thymidine, then [3H]thymidine
incorporation was measured using a beta counter. The results are
expressed as the mean ± SEM for triplicate wells during one
representative experiment from a total of five repeat
experiments.
