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Department of Immunology and Inflammation, Biogen, Cambridge, MA 02142
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence from a variety of studies suggests that manifestation of autoimmunity may involve dysregulated apoptosis. For instance, in vitro data suggest that PBMC of patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease characterized by autoantibody and immune complex-mediated tissue injury (2), exhibit an increased susceptibility toward apoptosis, (3, 4) or a defect in clearance of apoptotic cellular debris by scavenger macrophages (5). In general, cells that die as a consequence of normal cell turnover are rapidly phagocytosed by scavenger macrophages without inflammation or immune activation. However, apoptotic bodies are potential sources of self-Ag because they contain cellular components, including intracellular Ags that have translocated from their normal locations and clustered into membrane blebs (6). In fact, nucleosome, a natural self-Ag exposed to the immune system when cells undergo apoptosis, recently was identified as the major pathogen in (SWR x NZB)F1 (SNF1) lupus-prone mice (7), and interestingly, pathogenic Th clones isolated from lupus patients have been found to be specific for nucleosomal Ags (8). Furthermore, normal mice immunized with apoptotic but not viable cells produce autoantibodies (9). Thus, based on these observations it has been hypothesized that apoptotic bodies presented to self-reactive lymphocytes by competent APC promote autoimmunity. This may be especially relevant in settings of excessive cellular apoptosis whereby the scavenger system becomes overwhelmed and an atypical reservoir of self-Ag is established. Dendritic cells (DC) are critical APC for the initiation of T cell responses. Importantly, it recently was found that in addition to capturing and presenting exogenous Ag, immature as well as mature DC are capable of phagocytosing endogenous apoptotic cells and presenting self-Ag to T cells (10, 11, 12, 13), particularly when DC are exposed to high doses of apoptotic cells (14). However, despite the lines of evidence described above, there are no in vivo data to support the hypothesis that an increased frequency of apoptotic bodies is a critical factor underlying autoimmunity.
Given the important role of DC in Ag presentation, it is possible that disruption of normal DC regulation also may contribute to the loss of peripheral tolerance and generation of autoimmunity. DC, derived from bone marrow precursors, circulate in the blood and reside in almost every tissue. On encounter with Ag, immature DC migrate under the direction of chemokines (15) from peripheral tissues to the draining lymph nodes, where they potently stimulate Ag-specific CD4+ T cells. This is accompanied by an activated phenotype, which includes up-regulation of MHC class II and costimulatory molecules CD40, CD80, and CD86 (16). In addition, signals mediated by the interaction of CD154 on activated Th cells and the CD40 receptor on DC sustain their immunostimulatory capacity by prolonging DC survival and high MHC class II and costimulatory molecule expression (17). Therefore, it is possible to envision how imbalances in DC homeostasis and function may contribute to the persistence of autoimmunity.
To gain further understanding of the mechanisms underlying the promotion of autoimmunity and the loss of peripheral tolerance, we have investigated the cellular mechanisms maintaining autoimmunity in the SNF1 mouse model of SLE. SNF1 mice have been studied extensively and shown to resemble human SLE with nephritis occurring spontaneously in female mice (18). Previously, we have shown that autoimmunity in the SNF1 model is dependent on CD40/CD154 interactions (19). In this report, we reveal for the first time that the severity of autoimmune disease in the SNF1 model correlates with an increased frequency of splenic apoptosis and elevated numbers of splenic DC in vivo. The increase in DC numbers appears to be attributable to DC proliferation and enhanced DC migration associated with elevated splenic expression of secondary lymphoid chemokine (SLC). Importantly, the elevated levels of apoptotic cells, DC, and SLC were CD154 dependent. We discuss how these linked imbalances may form an autostimulatory loop that maintains autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SWR and NZB mice were purchased from The Jackson Laboratory (Bar Harbor, ME). SNF1 hybrids were bred in the animal facility at Biogen under conventional barrier conditions. Female SNF1 mice were used for all studies.
Mice undergoing mAb treatment received a single dose of 500 µg of anti-CD154 mAb (MR1; Ref. 20) or Armenian hamster IgG (HIgG) mAb Ha4/8-3.1, kindly provided by Dr. Donna Mendrick (Human Genome Sciences, Rockville, MD), once weekly for 6 wk, followed by a single injection of 500 µg monthly until age 11–12 mo. Mice began treatment at age 5.5 mo, at which time they exhibited moderate nephritis unless indicated otherwise. Assessment of renal disease was performed as described elsewhere (19).
Immunohistochemistry
Paraffin-embedded splenic tissue sections were used for H&E staining and for assessing apoptosis by TUNEL assay with an ApopTag kit (Intergen, Purchase, NY) according to the manufacturer’s recommendations, except that 1 µl of TdT was used and sections were counterstained with Nuclear Fast Red (Rowley Biochemical Institute, Danvers, MA). Frozen splenic tissue sections were fixed in acetone, air dried, and blocked for endogenous peroxidase with 0.09% H2O2 in methanol for 10 min. Sections were washed with TBS/.05%Tween 20 three times and then blocked in TBS/0.25%BSA/1.5% normal rabbit and goat sera plus Fc Block (BD PharMingen, San Diego, CA) for 1 h. Chemokine SLC was detected with biotin-conjugated anti-m6Ckine (R&D Systems, Minneapolis, MN) followed by streptavidin-HRP (Jackson ImmunoResearch, West Grove, PA), and visualized with the substrate 3,3'-diaminobenzidine (Sigma, St. Louis MO). Sections were counterstained with 0.05% Giemsa (Fluka, Buchs, Switzerland). B cells and DC were detected with anti-B220-FITC and anti-CD11c-biotin followed by streptavidin-PE, respectively (BD PharMingen). Images of sections stained with fluorochrome-tagged mAbs were captured by digitizing separate red and green images and were overlaid by using Adobe Photoshop (Adobe Systems, Mountain View, CA) software.
DC enrichment
Spleens were digested with collagenase type IV (Sigma) and erythrocytes were lysed with an ammonium chloride solution. The CD11c+ DC population was enriched with MACS CD11c microbeads and magnetic separation column (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s recommendations, except that the cell adherence step was eliminated. DC-enriched populations consisted of 23–54% DC, defined as CD11chigh.
Flow cytometry
Enriched DC and the splenocytes obtained from the flow-through
of the DC enrichment process (DC-depleted splenocyte fraction) were
used for determination of cell populations undergoing apoptosis. mAbs
directed against CD19, CD4, CD8
, Ly6-G/GR-1, and CD11c (BD
PharMingen) were used to identify B cells, T cells, granulocytes, and
DC, respectively. Anti-Annexin VFITC (BD
PharMingen) and 7-amino-actinomycin D (1 mg/ml; Calbiochem, La
Jolla, CA) uptake were used to identify apoptotic cells.
Phenotypic characterization of enriched CD11chigh
DC was performed with the following mAbs purchased from BD
PharMingen: anti-CD11c-biotin followed by
streptavidin-CyChrome, anti-CD8-FITC, and PE-conjugated
anti-MHC class II, CD40, CD80 (B7.1), CD86 (B7.2), CD13, CD95
(Fas), and CD54 (ICAM-1). Cells were incubated with 10 µg/ml Fc Block
(BD PharMingen) before addition of specific mAbs to inhibit FcR
binding. A total of 50,000–100,000 events were collected on a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed
with CellQuest software (BD Biosciences).
Proliferation analysis
To assess splenocyte proliferation, mice received i.v. 1.5 mg/10 g body weight of 5-bromo-2'-deoxyuridine (BrdU; Sigma) in PBS. Mice were sacrificed 1.5 h later, and splenic DC were enriched as described above. CD11chigh DC were assessed for BrdU incorporation by anti-BrdU-FITC or a control mouse IgG1-FITC mAb (BD PharMingen) and analyzed by flow cytometry.
DC migration studies
To obtain a pure population of splenic DC, pooled donor splenocytes from 21 to 25 healthy or 4 nephritic SNF1 mice were subjected to DC enrichment with anti-CD11c-coated magnetic beads as described above, visualized with anti-CD11c-biotin followed by streptavidin-PE, and sorted for CD11chigh cells on a MoFlo Cytometer (Cytomation, Ft. Collins, CO). Sorted DC were labeled with the cell-tracing reagent CFSE using the Vybrant CFDA SE Tracer kit (Molecular Probes, Eugene, OR) according to the manufacturer’s recommendations. CFSE becomes fluorescent on cleavage in live cells, allowing visualization under UV light. Healthy (ages 2–2.5 mo) and nephritic (ages 10–11.5 mo) SNF1 mice received 0.4–0.6 x 106 labeled DC i.v. from healthy or nephritic donors. After 22 h, spleens were harvested, snap frozen, and frozen sections were examined under UV light to determine the presence of donor DC. To compare the number of migrating DC among experimental groups, DC from six fields of view at x100 magnification were counted and totaled for each spleen section.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the finding that PBMCs from patients with SLE are more
susceptible to apoptosis in culture (3, 4), we asked
whether nephritic SNF1 mice exhibit increased
levels of programmed cell death in vivo. Splenic tissue sections from
untreated SNF1 mice at various stages of disease,
prenephritic (age 2 mo), moderately nephritic (age 5.5 mo) and severely
nephritic (age 10 or 11 mo), and a normal SWR parental control (age 11
mo) were used for TUNEL analysis. Relatively few apoptotic cells were
detected in the splenic white pulp of prenephritic
SNF1 and SWR control animals (Fig. 1
). However, we found that the frequency
of TUNEL+ cells in the splenic white pulp
progressively increased in SNF1 mice with
advancing age and disease severity (Fig. 1). Interestingly, 11-mo-old
SNF1 animals treated with a blocking
anti-CD154 mAb starting at age 5.5 mo displayed improved survival
of splenocytes as compared with control HIgG-treated or untreated mice
(Fig. 1), in addition to increased survival and inhibition of disease
progression (Ref. 19 and data not shown). When splenocytes
from the control HIgG-treated animals were further examined by
immunofluorescent staining and flow cytometry, we found a marked
increase in the percentage (7-fold) and number (3.4 x
106 cells) of apoptotic DC in the 11-mo-old
control HIg-treated SNF1 mice as compared with
the prenephritic animals (0.08 ± 0.01 x
106 cells), and smaller increases in the
percentage and number of apoptotic B cells (3-fold percent increase;
24.5 x 106 cells in HIgG-treated and
5.75 ± 0.15 x 106 cells in
prenephritic mice), T cells (3.5-fold percent increase; 2.4 x
106 cells in HIgG-treated and 0.6 ±
0.1 x 106 cells in prenephritic mice), and
granulocytes (3.6-fold percent increase; 0.8 x
106 cells in HIgG-treated and 0.07 ±
0.01 x 106 cells in prenephritic mice; Fig. 2). Subset analysis of splenocytes from
11-mo-old SNF1 mice treated with anti-CD154
mAb beginning at age 5.5 mo demonstrated that the increased percentages
and numbers of apoptotic DC (1.45 ± 0.65 x
106), B cells (16.7 ± 4 x
106), T cells (0.9 ± 0.4 x
106), and granulocytes (0.75 ± 0.35 x
106) were reduced with CD154 blockade (Fig. 2).
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Increased number of splenic DC in nephritic SNF1 mice correlates with disease severity and is reduced by anti-CD154 treatment
The increased frequency of apoptotic splenocytes in nephritic
SNF1 mice suggested a source of Ag for
presentation to self-reactive lymphocytes. Thus, we further
characterized the status of DC in these animals. Severely nephritic
animals (ages 10–12 mo) clearly exhibited splenomegaly (average spleen
weight, 1.2 ± 0.4 g, n = 7) as compared with
2- to 3-mo-old prenephritic SNF1 mice and
18-mo-old BALB/c mice (average spleen weight, 0.12 ± 0.03 g,
n = 6 and 0.13 ± 0.01 g, n =
3, respectively). Splenic hyperplasia in nephritic
SNF1 mice also was evidenced by total splenocyte
number, which increased from a mean of 78 ± 4.5 x
106 at age 2 mo to 239 ± 48 x
106 at age 11.5 mo, a 3.1-fold difference.
Histologic changes in the splenic architecture also were observed with
increasing spleen size, age, and disease severity (Fig. 3A). At age 2 mo, typical
areas of white pulp were visible surrounded by red pulp. However, by
age 5.5 mo, the white pulp was markedly expanded, and by age 11 mo, the
white pulp was so enlarged that there were few or no discernible areas
demarcating white and red pulp. In fact, the spleens of some mice
appeared to consist predominantly of white pulp. Changes within the
white pulp were further elucidated by immunofluorescent staining for B
cells and DC (Fig. 3B). Anti-B220 staining was used to
discriminate the B cell areas from the central T cell areas and showed
a normal pattern in healthy, prenephritic SNF1
mice. The DC pattern also was normal, with DC located either within the
T cell zone or in the marginal zone bridging channels. However, there
was a progressive increase in size of the white pulp, including a
striking increase in CD11c+ staining by age 5.5
mo. In addition, by age 11 mo, the normal localization of cells in the
white pulp was lost. The apparent detection of some
B220+CD11c+ cells
(appearing yellow) may reflect the colocalization of B cells and DC
in the diseased tissue or the appearance of a subpopulation of
CD11c+ B cells (21). Notably,
spleens from 11-mo-old mice treated with anti-CD154 beginning at
age 5.5 mo were reduced in size (average spleen weight, 0.37
± 0.1 g, n = 5) and exhibited smaller, more
organized white pulp areas as compared with 11-mo-old untreated or
HIgG-treated SNF1 mice, or age-matched parental
SWR mice (Fig. 3C).
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- and
CD11chighCD8+ DC
populations. As expected in healthy, prenephritic
SNF1 mice, the
CD11chighCD8- and
CD11chighCD8+ DC subsets
represented 1 and 0.28% of total splenocytes, respectively. We found a
progressive increase with age and disease severity in the percentage of
DC (Fig. 4). Interestingly, we also found
that the total DC number increased from a mean of 1.2 ± 0.1
x 106 in prenephritic mice to 8.7 ±
1.5 x 106 in nephritic animals. This
observation was disease-related, as normal BALB/c mice did not show an
increase in either total splenocyte or DC number with age (data not
shown). Furthermore, the percentage and number of splenic DC were
markedly reduced to normal or near normal levels by anti-CD154
treatment (Fig. 4).
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Phenotypic analysis of DC from nephritic SNF1 mice
Given the increased number of DC in the spleens of nephritic mice,
we asked if their phenotype was altered when compared with DC from
healthy animals. Mature splenic DC in normal mice are
CD11chigh and MHC class
IIhigh. These cells also express CD13
(22) and costimulatory molecules CD40, CD80, and CD86
(16), which are up-regulated with capture of Ag. Enriched
splenic DC subsets,
CD11chighCD8+ and
CD11chighCD8-, from
SNF1 mice aged 2.5 and 11–12 mo were analyzed by
flow cytometry to determine their surface phenotype. The
CD8+ DC subpopulation in both healthy and
nephritic SNF1 mice exhibited phenotypic markers
as described previously (16, 22), except that DC from
nephritic mice had slightly diminished CD95 expression as compared with
the same subpopulation from healthy animals (Fig. 5B). This CD95 decrease was
inhibited by long-term anti-CD154 treatment. The
CD8- DC subpopulation from nephritic mice
also displayed subtle changes, with decreased CD40, CD13, and CD54
expression and slightly increased CD95 expression as compared with DC
from prenephritic animals (Fig. 5A). The decreases in CD40,
CD13, and CD54 were inhibited by long-term anti-CD154 treatment,
although treatment did not prevent the increased CD95 expression we
observed in untreated nephritic animals.
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- DC in nephritic mice had an
unusual MHC class II expression pattern with 
50% of DC being MHC
class IIlow (Fig. 5
A), suggesting an
unusual accumulation of immature DC in addition to the mature DC in the
spleens of nephritic mice. To determine whether the MHC class
IIlow DC population could mature in vitro,
magnetic bead-enriched DC were cultured for 66 h with LPS,
anti-CD40 mAb, soluble CD154, or GM-CSF plus TNF-. Although all
cultures were activated as determined by up-regulated CD86 expression,
none of the culture conditions resulted in a shift to MHC class
IIhigh (data not shown), suggesting that a subset
of CD8- DC may be blocked in an immature
state. Accumulation of this DC subset in nephritic
SNF1 mice was not diminished by long-term
anti-CD154 treatment regardless of whether treatment began at age
5.5 mo (Fig. 5A), or before disease onset at age 3 mo (data
not shown). The nature and function of this population remains unclear.
Nevertheless, except for the appearance of a
CD8- MHC class IIlow
subset, DC from nephritic mice did not display a grossly distinct
phenotype. Splenic DC from nephritic SNF1 mice exhibit increased proliferation
Nephritic SNF1 mice clearly have an elevated
number of splenic DC. However, the mechanism for this increase is
unknown. One possibility is a proliferative expansion of DC. To test
this, prenephritic and nephritic SNF1 mice were
administered BrdU i.v. and their splenic
CD11chigh DC were analyzed for BrdU incorporation
by flow cytometry. DC from nephritic SNF1 mice
exhibited BrdU incorporation (Fig. 6, bottom left), whereas DC from prenephritic mice had little
or no detectable BrdU incorporation (Fig. 6, top left).
Thus, in contrast to splenic DC from healthy animals, the DC from
nephritic SNF1 mice are actively proliferating.
Interestingly, long-term anti-CD154 mAb treatment did not effect
the proliferative status of DC from nephritic mice because 11-mo-old
SNF1 mice that received anti-CD154 mAb
continuously from age 5.5 mo had an anti-BrdU staining profile
(Fig. 6, bottom right) that was comparable to that of
control HIg-treated (Fig. 6, top right) and untreated mice
(data not shown).
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The increased proliferative activity of splenic DC in nephritic
SNF1 mice cannot be the sole mechanism for the
elevated number of DC because anti-CD154 mAb treatment, which
resulted in near normalization of the splenic DC number, had no
antiproliferative effect. Therefore, we hypothesized that in nephritic
mice there is enhanced DC migration to the spleen. This could be
attributable to the splenic microenvironment of nephritic animals
and/or factors intrinsic to their DC. To test this hypothesis, we
compared the ability of purified CD11chigh
splenic DC from healthy SNF1 mice (prenephritic
DC) and nephritic SNF1 mice (nephritic DC)
labeled with CFSE to migrate to the spleen after injection of each
population into both healthy and nephritic SNF1
animals. Migration to recipient spleens was visualized by fluorescence
microscopy. Very few "prenephritic" (12 ± 6) and
"nephritic" (11 ± 0) DC were detected in the spleens of
prenephritic SNF1 recipients (Fig. 7, A and C). In
contrast, numerous prenephritic (100 ± 10) DC and nephritic
(87 ± 12) DC were clearly visible in the spleens of nephritic
recipients (Fig. 7, B and D). Thus, migration of
DC to spleens of nephritic mice is 9-fold greater than to spleens of
healthy animals. Splenic tissue sections from these mice were also
stained for B220+ B cells to provide orientation
within the spleen, and this revealed that the vast majority of labeled
DC were localized within the splenic white pulp (data not shown). These
data suggest that nephritic SNF1 mice exhibit a
unique splenic microenvironment that promotes DC recruitment.
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Given that the splenic microenvironment of nephritic mice appears
to foster DC recruitment to the spleen, we speculated that the levels
of DC homing chemokines such as SLC and ELC (15)
might be elevated in the spleens of these animals. Expression of SLC,
produced by splenic stromal cells within the T cell-rich area of the
white pulp and by DC, was assessed immunohistochemically in
prenephritic mice, in 10- to 11-mo-old nephritic mice, and in 12-mo-old
SNF1 mice treated with anti-CD154 mAb from
age 5.5 mo. Our studies reveal increased SLC expression in the T cell
areas of spleens from nephritic SNF1 mice as
compared with healthy, prenephritic animals (Fig. 8, top). Interestingly,
12-mo-old SNF1 mice that received long-term
anti-CD154 mAb treatment had levels of splenic SLC that resembled
more closely those in prenephritic mice (Fig. 8, bottom).
Thus, our data demonstrate that SLC expression is elevated in spleens
of diseased SNF1 animals and that these
abnormally high SLC levels are dependent on CD154. Furthermore, they
suggest that anti-CD154 mAb treatment reduces splenic DC numbers in
nephritic animals, at least in part, by reducing expression of the DC
homing chemokine, SLC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increased frequency of apoptosis in the spleens of nephritic SNF1 animals supports the hypothesis that the increased apoptosis observed in lupus patient PMBC in vitro occurs in vivo. Increased apoptosis in vivo may reflect an intrinsic defect in cells that predisposes them to die at a higher rate and/or a defect in apoptotic cell clearance. Our studies in SNF1 mice reveal a marked increase in the proportion of apoptotic DC and a smaller increase in the proportion of apoptotic B cells, T cells, and granulocytes in nephritic animals. Thus, it is unlikely that this increased apoptosis is attributable to a generic defect in cell death machinery. Rather, given the immune activation in this model as evidenced by splenic hyperplasia and our observation that white pulp expansion and elevated apoptosis are dependent on CD154, we speculate that the increased frequency of apoptotic cells in nephritic SNF1 mice results from CD154-dependent cell activation and subsequent activation-induced cell death. The CD40/CD154 pathway provides critical signals for the activation, maturation, and survival of many cell types (reviewed in Ref. 25). Notably, CD154 is known to be dysregulated in SLE patients (26, 27, 28) and lupus-prone mice (29), leading to elevated and prolonged CD154 expression. Because the CD40/CD154 pathway has been demonstrated to provide DC and B cell survival signals, inhibition of apoptosis through blockade of this pathway with anti-CD154 may seem paradoxical. However, under certain conditions, signaling through CD40 on B cells has been shown to result in an increased susceptibility to apoptotic cell death (30). This along with data from previous reports involving other receptors of the TNF family (31, 32) suggest that the outcome of receptor signaling can vary depending on the combination of surface receptors that are engaged and the intracellular and extracellular environments. The mechanism for splenic DC death is just beginning to be elucidated. Although some investigators have speculated that the CD95/FasL pathway provides a mechanism for DC death, recent reports suggest that splenic DC are resistant to CD95/FasL-regulated apoptosis (33). Currently, two apoptotic pathways for DC have been identified: apoptosis via MHC class II cross-linking (34) and ATP-mediated apoptosis via the P2Z/P2X7 receptor (35). It currently is unknown whether one or both of these mechanisms contribute to the observed cell death in SNF1 mice. Regardless of the mechanism, our results establish for the first time that CD154-mediated signals regulate the abundance of self-Ag in this lupus-like disease setting.
DC are recognized as key players in central tolerance in the thymus,
and subtypes of DC may play a role in peripheral tolerance
(36). However, DC also are recognized for their critical
role in initiating robust adaptive immune responses to exogenous as
well as endogenous Ag. Importantly, our studies revealed a 7.3-fold
increase in the number of DC in the spleens of nephritic
SNF1 mice, with the greatest increase (8.3-fold)
occurring within the CD8- subset.
Approximately 50% of these DC have a mature phenotype. The importance
of the preferential increase in CD8- DC is
unclear because both the CD8+ and
CD8- DC subsets have been found to possess
near equivalent capability for Ag uptake in vivo (37).
However, it has been suggested that each DC subset regulates T cell
responses differently, and recent data indicate that
CD8- DC induce a Th2-type response, whereas
CD8+ DC induce a Th1-type response
(37, 38, 39). In addition, CD8- DC
were found recently to exclusively express two newly identified genes,
CIRE and FIRE (37). As the function of these and other
novel DC-specific gene products are elucidated, an understanding of the
roles of the different DC subsets in maintaining autoimmunity will
emerge.
It was apparent from our results that the elevated splenic DC number in
nephritic mice may result, in part, from proliferation. However, the
ability of anti-CD154 treatment to significantly reduce DC number
without affecting their proliferation and our adoptive transfer studies
suggest that the CD154-dependent increase was likely regulated at the
level of migration to the spleen. Thus, we have demonstrated that the
splenic microenvironment of nephritic animals uniquely promotes
recruitment of DC derived from healthy or nephritic animals, whereas we
were unable to detect a difference in trafficking ability intrinsic to
the DC. We have further shown increased expression of SLC in splenic
tissue from nephritic animals that was CD154 dependent. It is difficult
to discern whether the CD154-dependent increase in SLC expression is
attributable to the increased cellularity of the spleen or whether
CD154-mediated signals also increase SLC production on a per cell
basis. Additional studies are required to quantify the differences in
SLC expression and better address the ability of CD154 to directly
regulate SLC production. Nevertheless, our results suggest that splenic
DC migration in autoimmune SNF1 mice is regulated
by CD154, potentially through its regulation of SLC. A link to SLC may
also be important because chemokines, in addition to directing cell
migration, can provide positional cues directing the localization of
different cell types within lymphoid organs. This concept is supported
by precedence with another TNF family ligand, LT
, which provides
critical cell positioning cues in the spleen through its regulation of
chemokines BLC, SLC, and ELC (40). Thus, we speculate that
the disorganization observed in the splenic white pulp of
SNF1 mice, which was prevented by anti-CD154
treatment, may be mediated by a CD154/chemokine axis.
Our findings reported here, along with previous studies demonstrating hyperexpression of CD154 in lupus settings, show that three critical immune system components are altered in lupus-like autoimmune disease: 1) an abundant source of self-Ag, 2) an abundance of APC, and 3) an elevated level of CD154-mediated costimulatory signals. Although these results do not speak to disease initiation, we show that imbalances in these components are ultimately linked to each other and correlate with disease progression. As such, they have the capacity to form a stimulatory loop that perpetuates self-reactivity in vivo. Of course, we cannot rule out that other costimulatory molecules are also an integral part of this progression.
In summary, our studies delineate peripheral mechanisms likely underlying persistent autoimmunity in a mouse model of SLE. We demonstrate altered regulation of apoptosis and DC homeostasis that appear to be CD154 pathway dependent. To determine whether these observations are directly linked to CD154 or indirectly linked through activation of additional costimulatory pathways and/or chemokine or cytokine production requires further investigation because any mechanism which interferes with pathogenic T cell, B cell, or APC function may be effective in inhibiting disease progression. These findings may be relevant to the development of therapeutic strategies for the treatment of autoimmune disorders.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: FasL, Fas ligand; DC, dendritic cell; SLC, secondary lymphoid chemokine; SLE, systemic lupus erythematosus; HIg, hamster Ig, BrdU, 5-bromo-2'-deoxyuridine.
Received for publication February 22, 2001. Accepted for publication May 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359. receptor II-mediated apoptosis of cytotoxic T lymphocytes. J. Exp. Med. 188:1391.+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587./ and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403.This article has been cited by other articles:
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S. Wan, C. Xia, and L. Morel IL-6 Produced by Dendritic Cells from Lupus-Prone Mice Inhibits CD4+CD25+ T Cell Regulatory Functions J. Immunol., January 1, 2007; 178(1): 271 - 279. [Abstract] [Full Text] [PDF]
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H.-K. Kang, M. A. Michaels, B. R. Berner, and S. K. Datta Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets J. Immunol., March 15, 2005; 174(6): 3247 - 3255. [Abstract] [Full Text] [PDF]
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K. Fujio, A. Okamoto, H. Tahara, M. Abe, Y. Jiang, T. Kitamura, S. Hirose, and K. Yamamoto Nucleosome-Specific Regulatory T Cells Engineered by Triple Gene Transfer Suppress a Systemic Autoimmune Disease J. Immunol., August 1, 2004; 173(3): 2118 - 2125. [Abstract] [Full Text] [PDF]
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W.-K. Ip and Y.-L. Lau Distinct Maturation of, but Not Migration between, Human Monocyte-Derived Dendritic Cells upon Ingestion of Apoptotic Cells of Early or Late Phases J. Immunol., July 1, 2004; 173(1): 189 - 196. [Abstract] [Full Text] [PDF]
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 S. Rajagopalan, E. C. Somers, R. D. Brook, C. Kehrer, D. Pfenninger, E. Lewis, A. Chakrabarti, B. C. Richardson, E. Shelden, W. J. McCune, et al. Endothelial cell apoptosis in systemic lupus erythematosus: a common pathway for abnormal vascular function and thrombosis propensity Blood, May 15, 2004; 103(10): 3677 - 3683. [Abstract] [Full Text] [PDF]
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 J Yazdany and J Davis The role of CD40 ligand in systemic lupus erythematosus Lupus, May 1, 2004; 13(5): 377 - 380. [Abstract] [PDF]
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L. Gorelik, A. H. Cutler, G. Thill, S. D. Miklasz, D. E. Shea, C. Ambrose, S. A. Bixler, L. Su, M. L. Scott, and S. L. Kalled Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function J. Immunol., January 15, 2004; 172(2): 762 - 766. [Abstract] [Full Text] [PDF]
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