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Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1
2, it is unclear whether other homeostatic chemokines have
this property. In this work we show that ectopic expression in
pancreatic islets of CCL19 leads to small infiltrates composed of
lymphocytes and dendritic cells and containing high endothelial venules
and stromal cells. Ectopic CXCL12 induced small infiltrates containing
few T cells but enriched in dendritic cells, B cells, and plasma cells.
Comparison of CCL19 transgenic mice with mice expressing CCL21
(secondary lymphoid tissue chemokine) revealed that CCL21 induced
larger and more organized infiltrates. A more significant role for
CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found
to be present in lymph nodes and spleen at much higher concentrations
than CCL19. CCL19 and CCL21 but not CXCL12 induced LT12
expression on naive CD4 T cells, and treatment of CCL21 transgenic mice
with LTR-Fc antagonized development of organized lymphoid
structures. LT12 was also induced on naive T cells by the
cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12
are sufficient to mediate cell recruitment in vivo and they indicate
that LT12 may function downstream of CCL21, CCL19, and IL-2
family cytokines in normal and pathological lymphoid tissue
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphoid tissue organization is promoted by a subset of the chemokine
family, sometimes called homeostatic or lymphoid chemokines because of
their constitutive expression in preformed lymphoid tissues (5, 6). These include CXCL13 (B lymphocyte chemoattractant), the
ligand of CXCR5; CCL19 (EBV-induced molecule 1 ligand chemokine) and
CCL21 (secondary lymphoid tissue chemokine), ligands for CCR7; and
CXCL12 (stromal cell-derived factor 1), the ligand of CXCR4
(7). CXCL13 is made by stromal cells in B cell follicles
and is required for homing of B cells to the follicular compartment
(8). Expression of CXCL13 in pancreatic islets of
transgenic mice was found not only to cause B cell recruitment but also
to lead to development of ectopic lymph node-like structures containing
T cell areas, with high endothelial venules
(HEVs)4 and stromal
cells (9). Development of these structures was dependent
on signaling by lymphotoxin (LT)12. These observations indicated
that CXCL13 could activate a LT-dependent process of lymphoid
neogenesis (9). Naive B cells have been shown to be an
important source of LT12 and more recently it was established
that CXCL13 can up-regulate LT12 on B cells (8, 10).
Activated T cells and NK cells can also express LT12 (11, 12). Studies in CXCR5-deficient and CXCL13-deficient mice have
confirmed that these molecules are important in the development of many
secondary lymphoid organs (8, 13), and LT12 and its
receptor, LTR, are also critical for normal development of all
secondary lymphoid organs (10).
CCR7 and its ligands function to guide naive T cells and maturing dendritic cells (DC) into T zones of secondary lymphoid organs (6). CCL19 and CCL21 are both constitutively expressed by stromal cells within lymphoid T zones and CCL21 is expressed by HEVs and, at lower levels, by lymphatic endothelium (14, 15, 16). In addition to stromal cells, CCL19 expression is found in DCs (16, 17, 18, 19, 20). Recent evidence suggests that CCL19 protein can be translocated onto the lumen of HEVs (21). In vitro, CCL19 and CCL21 induce chemotaxis of mature DCs, naive and activated T cells, and, to a lesser degree, B cells. Furthermore, ectopic expression of CCL21 in the pancreatic islets was sufficient to cause infiltration by T cells, B cells, and DCs as well as to induce HEVs (22, 23). However, to what extent these effects reflect the direct action of CCL21 vs induction of downstream events was not clear, and the relative in vivo activities of CCL19 and CCL21 remain unknown. Both CCL19 and CCL21 have been found to be up-regulated at sites of chronic inflammation (24, 25, 26, 27, 28, 29).
The CXCL12/CXCR4 ligand/receptor pair is critical in bone marrow hematopoiesis and plays important roles in gut vasculogenesis and heart and brain development, and deficiency in either the receptor or the ligand causes perinatal lethality (Ref. 30 and references therein). Consistent with these diverse functions, CXCL12 (stromal cell-derived factor 1) is expressed broadly, including expression by stromal cells within bone marrow and in many epithelial tissues. Within secondary lymphoid tissues, there is expression in the splenic red pulp, in lymph node medullary cords, and in the subepithelial region of tonsil (31, 32). In vitro, CXCL12 is a chemoattractant for naive B cells and T cells as well as DCs and plasma cells (Refs. 30 and 31 and references therein). Recently, CXCR4-deficient plasma cells were shown to localize aberrantly in the spleen and failed to accumulate appropriately in the bone marrow (31). CXCL12 is strongly up-regulated in the joint synovium of rheumatoid arthritis patients and may also contribute to skin inflammatory responses (33, 34, 35).
The strong in vitro chemotactic activities of CCL19, CCL21, and CXCL12
for T cells and DCs, together with their frequent expression at sites
of inflammation, led us to generate transgenic mice to examine the in
vivo chemotactic and nonchemotactic functions of these chemokines.
Using the rat insulin promoter (RIP) system (36), we
demonstrate that all three chemokines are able to induce lymphoid
structures within the pancreas, but we find that these structures show
striking differences in size, cellular composition, and organization.
The differences are shown to correlate with chemokine protein
concentration and the propensity to induce LT12 on naive T cells.
We also show that members of the IL-2 cytokine family induce LT12
expression on naive T cells and suggest that they also may activate a
LT12-dependent pathway of lymphoid tissue formation. These
results have implications for our understanding of lymphoid tissue
organization, chronic inflammatory responses, and lymphomagenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate transgenic mice, the full-length cDNA of murine
CCL19, CCL21-leu (CCL21b), and CXCL12 was cloned into a ClaI
site of the 10-kb RIP7 promoter construct (9). Linearized
constructs were injected into B6D2F1 oocytes and transgenic mice
backcrossed to C57BL/6 for one to five generations. Seven lines of
RIP-CCL19 (transgene copy number estimated as 12 and 76 for lines I and
II, respectively), three lines of RIP-CCL21 (13, three, and eight
copies for lines I, II, and IV, respectively), and four lines of
RIP-CXCL12 (34, 31, nine, and one copies for lines I, II, III, and IV,
respectively) were established and characterized. Homogenous expression
in all cells was obtained with all mouse lines except RIP-CCL21
line IV and RIP-CXCL12 line I, where expression was restricted to a
small subset of cells. Most results presented were obtained with
RIP-CCL19 line II, RIP-CCL21 line II, and RIP-CXCL12 line II.
RIP-CXCL13 mice have been previously described (9). None
of the transgenic mouse strains developed obvious symptoms of diabetes.
Mice were screened by PCR using the following primers:
5'-CAACCCTGACTATCTTCCAG (RIP7, forward),
5'-GAGATGATAGTGGCTTCAGGCAG-3' (CXCL13, reverse),
5'-CTACTAATCGATCCAGAGTGATTCACATCTC-3' (CCL19, reverse),
5'-CTACTAATCGATGGACCGTGAACCACCCAG-3' (CCL21, reverse), or
5'-TAGTAGATCGATGTCCTTTGGGCTGTTGTGC-3' (CXCL12, reverse).
Some adult RIP-CCL21 mice received i.p. injections of 100 µg soluble
human LFA3-Fc or murine LTR-Fc fusion protein (kindly provided by J.
Browning, Biogen, Cambridge, MA) two times a week as previously
described (37). Tissues were harvested after 20 days.
Nonobese diabetic (NOD) females were from Jackson ImmunoResearch
Laboratories (West Grove, PA).
Immunohistochemistry
Mice were euthanized with CO2 and tissues were removed and frozen in OCT compound (Miles, Elkhart, IN). Cryostat sections (10 µm) were collected on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA), dried overnight, fixed for 10 min in acetone (4°C), and stained with reagents and procedures described previously (9). Chemokine stainings were done in four steps: blocking with 0.1% BSA and 4% normal mouse and donkey serum, application of polyclonal goat sera to CCL19, CCL21 (R&D Systems, Minneapolis, MN), or CXCL12 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) and streptavidin-ABC (Vector Laboratories, Burlingame, CA). Plasma cells were detected using a rat anti-mouse IgM-biotin (Caltag Laboratories, Burlingame, CA) followed by streptavidin-AP (Vector Laboratories).
Quantification of histology
Every fifteenth 10-µm section from the pancreas was counterstained with hematoxylin and the number of islets and the number of mononuclear cells per islet were counted. Infiltrates were scored as small (5–30 cells), medium (31–300 cells), and large (>300 cells). Islets with fewer than five mononuclear cells were scored as noninfiltrated. This procedure was applied for 80–160 islets per pancreas.
Western blot analysis
Whole spleen or a pool of mesenteric and peripheral lymph nodes was homogenized in ice-cold lysis buffer (120 mM NaCl, 50 mM Tris-HCl (pH 8), 1 mM EDTA, 6 mM EGTA, 1% Nonidet P-40) containing protease inhibitors (1 mM PMSF, 1 mM benzimidine, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). For pancreas, the protease inhibitor TLCK was also added at 50 µg/ml. Debris was removed by centrifugation and supernatant was either used as whole lysate or immunoprecipitated using heparin-Sepharose as previously described (16). Detection was with goat anti-mouse CCL19, CCL21, CXCL13 (R&D Systems), or CXCL12 (Santa Cruz Biotechnology) followed by anti-goat HRP (Jackson ImmunoResearch Laboratories); development was with ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ). Specificity was controlled by showing lack of reactivity of the CCL19-, CCL21-, and CXCL13-specific Abs with lysates from CCL19/CCL21-deficient (plt/plt) mice (16, 38) or CXCL13-deficient mice (8). Titrations of recombinant chemokines (R&D Systems) were used to quantify the amount of chemokines in tissues. Concentrations of chemokines in tissues was calculated by dividing the amount of chemokine per total tissue with the average weight for pancreas (90 mg), spleen (90 mg), and pooled lymph nodes (40 mg).
Quantitative RT-PCR analysis
PCR primers and probes for CCL19, CCL21, and hypoxanthine phosphoribosyltransferase (HPRT) were as previously described (16). CCL19 primers were specific for the functional CCL19 transcript (CCL19-atg), with a new 3' primer (5'-cttccgcatcattagcaccc-3'). The CCL21 primers detect total CCL21 transcript levels and do not distinguish between CCL21-ser (CCL21a) and CCL21-leu (CCL21b) transcripts (39). Quantitative RT-PCR was performed on an ABI 7700 sequence detection instrument (TaqMan; PE Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. The relative efficiency of CCL19 and CCL21 mRNA amplification was established using various amounts of expressed sequence tag plasmids encoding CCL19-atg (clone AA444730) or CCL21-ser (clone AW987545.1). After correction for the difference in amplification efficiency the expression of CCL19 and CCL21 in mRNA samples was quantified using HPRT as a reference. The specificity of the primers used to amplify CCL19 and CCL21 have previously been controlled by showing a >4000-fold lower amplification in samples from plt compared with wild-type mice (16). Lymph node pool was composed of mesenteric and peripheral nodes.
In vitro LT12 induction and flow cytometry
Spleen cell suspensions were prepared, RBCs were lysed, and
cells were preincubated for 14–17 h in flat-bottom 96-well plates and
complete RPMI medium containing 10% FCS to allow adjustment to in
vitro conditions. For pretreatment with pertussis toxin (Ptx), cells
were incubated for 2 h with 200 ng/ml toxin. The indicated
concentration of recombinant chemokines or cytokines (R&D Systems) was
added for 6 h at 37°C. Cells were placed on ice, washed, and
stained for surface LT12 using LTR-Fc fusion protein
(37). In brief, cells were pretreated with FcR blocking Ab
(2.4.G2; BD PharMingen, San Diego, CA), 0.5% normal mouse and rat
serum, and, if indicated, anti-LT blocking Ab (BBF6). LTR-Fc
was added and detected using either F(ab')2 goat
anti-human Ig-PE (Jackson ImmunoResearch Laboratories) or
biotinylated goat anti-human IgG (Jackson ImmunoResearch
Laboratories), both of which were pretreated for 30 min with 4% normal
mouse and rat serum. Finally, FITC-conjugated anti-CD69 (BD
PharMingen), PE-conjugated anti-CD4 and anti-CD8 (Caltag
Laboratories), PerCP-conjugated anti-B220 (BD PharMingen), and
streptavidin-allophycocyanin were added (Molecular Probes,
Eugene, OR). Cells were analyzed using a four-color FACSCalibur (BD
Biosciences, Mountain View, CA) and FlowJo software (BD
Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the in vivo activities of CCL21 and CCL19, a total of
three lines of RIP-CCL21 and seven lines of RIP-CCL19 transgenic mice
were established and characterized. Histological analysis of pancreas
tissue from each of the transgenic mouse lines showed detectable
chemokine protein expression within cells of pancreatic islets
(Fig. 1
A and data not shown).
Nontransgenic pancreas showed a low level of CCL21 expression that was
limited to lymphatic endothelium, as previously reported
(9), and no detectable CCL19 (data not shown). Pancreatic
islets of RIP-CCL21 transgenic mice were often infiltrated by large
numbers of CD4+ and CD8+ T
cells, IgD+ B cells, and CDIIc+ DCs
(Fig. 1A), confirming two previous reports on independently
generated RIP-CCL21 transgenic mouse lines (22, 23). No
evidence for infiltration by Mac-I+ or
MOMA-1+ macrophages was obtained. Interestingly,
CCL19 induced fewer and smaller infiltrates that were often associated
with large blood vessels (Fig. 1A). This observation is
representative for all seven RIP-CCL19 transgenic lines. These small
cellular accumulations were rare in young mice and increased in
frequency in older animals (Fig. 2A; see also Fig. 4C). Flow cytometric analysis of infiltrates in pancreas of
old RIP-CCL19 and RIP-CCL21 mice showed a similar composition, with B
cells representing the largest population and CD4 T cells being more
frequent than CD8 T cells (Fig. 2B). Although CCL19 was
sufficient to attract the same cell types as CCL21, CCL19-induced
infiltrates were less organized, with T cells, B cells, and DCs
frequently being interspersed (Fig. 1A). Besides attracting
various hematopoietic cell types, both chemokines were sufficient to
cause development of HEVs, characterized by thick endothelium and
expression of peripheral lymph node addressin (PNAd) and mucosal
addressin cell adhesion molecule (MAdCAM) (Fig. 1B and data
not shown). Surprisingly, CCL21 and CCL19 protein were not detected on
HEVs within the infiltrate of the respective mouse lines (Fig. 1A and data not shown). Both chemokines also caused variable
but reproducible appearance of gp38+,
BP3+, and ER-TR7+ stromal
cell networks within the infiltrates (Fig. 1B and data not
shown). Consistent with the larger infiltrate size and more defined
organization of T and B zones, CCL21 showed a higher efficiency than
CCL19 in inducing HEVs and stromal cell networks (Fig. 1B).
Neither of the two mouse models showed evidence for development of
CD35+ follicular DCs or CXCL13-expressing stromal
cells (data not shown).
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C). Another important difference between the
infiltrates induced by CCR7 and CXCR5 ligands was that CCL21, and to a
lesser degree CCL19, induced infiltrates showing an invasive phenotype
with a ragged boundary between accumulating T cells and islet cells and
the frequent presence of individual T cells within the islet cell mass
(Fig. 2C and data not shown). This intraislet invasion was
only rarely observed in RIP-CXCL13 transgenic mice, where large numbers
of B cells are found to accumulate next to clusters of islet cells
without mixing of islet cells and lymphocytes (Fig. 2C). An
analysis of the autoimmune infiltrates that form in islets of NOD mice
has previously shown the presence of CCL21 transcripts
(26). By immunohistochemistry, we were able to detect
CCL21 protein on vessels and stromal cells within infiltrates of
prediabetic NOD mice (Fig. 2D). A fraction of the
infiltrates in NOD mice shows intraislet invasiveness
(40), a feature possibly important in islet cell
destruction. However, no signs of diabetes were observed in RIP-CCL21
or RIP-CCL19 transgenic animals up to 12 mo of age, establishing that
chemokine-mediated islet invasion by T cells is not sufficient to cause
disease.
To examine whether CCL19 and CCL21 may function in a synergistic manner
in promoting ectopic lymphoid tissue development, we generated mice
coexpressing CCL19 and CCL21 by intercrossing RIP-CCL19 and RIP-CCL21
transgenic mice. No increase of infiltrate size in double transgenic
compared with CCL21 single transgenic mice was detected by
hematoxylin staining (Fig. 2A). Similarly,
immunohistochemical analysis showed little difference in the
composition and orientation of the pancreatic infiltrates in double vs
CCL21 single transgenic mice (data not shown).
Differential expression of CCL21 and CCL19 in transgenic pancreas and secondary lymphoid organs
To investigate the basis for the differential efficiency of CCL21
vs CCL19 in induction of infiltrates in RIP transgenic mice, we first
measured the amount of the respective chemokines present within the
pancreas. Western blot of lysates from RIP-CCL21 pancreas revealed
large amounts of CCL21 protein (2–20 ng per milligram of pancreas),
while the amounts present in nontransgenic pancreas were below
detection (Fig. 3, A and
D). Assuming similar detection of endogenously expressed
recombinant CCL19, we estimate this chemokine was present only at very
low levels (0.01–0.2 ng/mg) in pancreatic lysates from RIP-CCL19
transgenic mice (Fig. 3, A and D). This
100-fold difference in CCL19 and CCL21 protein levels was
consistently observed in pancreas tissue from mouse lines derived from
at least two founders of each type (data not shown). Analysis of
chemokine mRNA levels in the two types of transgenic mice by
quantitative PCR revealed a difference of <10-fold (Fig. 3E), consistent with the use of identical promoter and
enhancer regions in the transgenic constructs.
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B). When chemokines and other basic proteins were
enriched from the lysates by precipitation with heparin-Sepharose,
CCL19 became detectable (Fig. 3C). Our analysis indicates
>100-fold higher levels of CCL21 protein compared with CCL19 in both
spleen and lymph nodes (Fig. 3D). By contrast, CCL19 and
CCL21 mRNA levels in spleen and lymph nodes differed by <5-fold (Fig. 3F). Therefore, the large difference in protein levels in
lymphoid tissues and in the transgenic system indicates that CCL19
protein accumulates in tissues less efficiently than CCL21 protein. A
similar quantitative analysis of CXCL13 in lymphoid organs revealed
protein expression levels that are a few fold above CCL19 but
50-fold lower than CCL21 (Fig. 3, C and D).
These results are consistent with protein staining data on histological
sections of secondary lymphoid organs where CCL19 is undetectable and
CCL21 is more easily detectable than CXCL13 (Refs. 8, 9 ,
and 16 and data not shown). Ectopic expression of CXCL12 induces DC and plasma cell accumulation
Similar to CCL19 and CCL21, CXCL12 attracts T and B cells as well
as DCs in vitro. To investigate CXCL12 function in vivo, RIP-CXCL12
transgenic mice were generated. Expression of CXCL12 in pancreatic
islets caused only small, occasional infiltrates to develop, although
these became more frequent in older mice (Fig. 4, A and C). Unlike
the small infiltrates in RIP-CCL19 transgenic mice, the CXCL12-induced
cell accumulations consisted mainly of naive B cells with only small
numbers of T cells. Strikingly, however, the CXCL12-induced infiltrates
contained large proportions of DCs and plasma cells (Fig. 4A). Plasma cells were only occasionally found in
infiltrates of RIP-CCL21 (Fig. 4B), RIP-CCL19, and
RIP-CXCL13 transgenic mice (data not shown). In some cases, these
infiltrates were associated with stromal cell networks and small
vessels expressing HEV markers (data not shown). We examined a total of
four RIP-CXCL12 mouse lines and found similar infiltrations in a second
line, whereas two lines showed minimal CXCL12 expression and no
detectable infiltration. CXCL12 protein levels in the highest
expressing mouse line were estimated to be 1–2 ng chemokine per
milligram of pancreas (data not shown). This amount is 5- to 10-fold
below the level of CCL21 in the pancreas of RIP-CCL21 mice, 4- to
8-fold above the level of CXCL13 in RIP-CXCL13 mice, and 10- to 40-fold
above the level of CCL19 in RIP-CCL19 mice (Fig. 3D).
Comparing the four lymphoid chemokine transgenic mouse models, only a
partial correlation between chemokine expression level and infiltration
frequency can be observed. CCL21 and CXCL13 induce a high frequency of
infiltrates of all sizes in young mice, while mice expressing CCL19 and
CXCL12 only develop small and medium-size infiltrates, and these only
become prominent when the animals are more than 4 mo of age (Fig. 4C).
Chemokine-mediated LT12 induction on T cells
In addition to quantity and relative activity in promoting
chemotaxis, a further property of chemokines influencing their activity
in promoting ectopic lymphoid tissue development is their ability to
induce LT12 expression (8). Surprisingly, although
naive T cells have not been reported to express LT12, we found
that CD4 and CD8 T cells from blood constitutively expressed very low
amounts of membrane LT12 (Fig. 5A). The specificity of
LTR-Fc for LT12 was confirmed by the lack of staining on
LT-deficient T cells (data not shown) and by the background level of
staining in the presence of a LT blocking Ab (Fig. 5A).
In contrast to B cells, LT12 expression was not increased on T
cells in lymph nodes (Fig. 5A) or spleen (data not shown),
suggesting that lymphoid tissue chemokines may not be involved in
inducing LT12 expression on naive T cells. However, when
recombinant CCL21 was added to splenocytes in vitro, LTR-Fc ligands
on CD4 T cells were strongly up-regulated within 6 h (Fig. 5B). Addition of an LT blocking Ab reduced staining to
levels equivalent to nontreated cells (Fig. 5B). When
splenocytes were pretreated with Ptx before adding CCL21, up-regulation
of LT12 was prevented, indicating involvement of
Gi protein-coupled signals downstream of CCR7.
When other T cell chemoattractants were tested, CCL19 but not CXCL12
induced LT12 up-regulation on naive CD4 T cells (Fig. 5B). To compare CD4 and CD8 T cells and B cells for their
sensitivity to chemokine-mediated induction of LT12, a titration
analysis was performed using all four previously described chemokines.
Surprisingly, concentrations as low as 0.1 nM CCL19 and 1 nM CCL21 were
sufficient to induce LT12 expression on CD4 T cells (Fig. 5C). In contrast, CD8 T cells showed only a very modest
induction of LT12, even at high concentrations of CCL19 and CCL21
(Fig. 5D). CXCL12 failed to cause notable LT12
expression on T cells (Fig. 5, C and D) but was
capable of promoting expression on B cells (Fig. 5E), as
previously observed (8). Interestingly, the concentrations
of CXCL13 required for LT12 induction on B cells (Fig. 5E) were >10-fold higher than the amounts of CCL21 needed
to promote strong LT12 up-regulation on CD4 T cells.
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R-Fc treatment of RIP-CCL21 mice causes loss of HEV and
stromal cells
To test whether CCL21-induced pancreatic infiltrates were
dependent on signals by the LT12-LTR pathway, we treated
RIP-CCL21 transgenic mice for 3 wk with LTR-Fc or with a control
protein (hLFA3-Fc). Histological analysis showed only a weak effect of
LTR-Fc treatment on infiltrate size, composition, and orientation
(Fig. 6A). Strikingly,
however, administration of LTR-Fc strongly reduced the expression of
the adhesion molecules PNAd and MAdCAM on HEVs within infiltrated
islets (Fig. 6B). Filamentous stromal cell networks,
identified by gp38, BP3, and PNAd staining, were also greatly affected
by this treatment (Fig. 6B). Our attempts to identify the
cell population expressing LT12 in infiltrates of RIP-CCL21
pancreas were not successful, most likely due to the enzymatic digest
required for the isolation of infiltrating cells and the low
sensitivity of the detection of surface LT12. In summary, in this
experimental setting LT12 appears to play a specific role
downstream of CCL21 that includes the induction or maintenance of
activated endothelial cells and stromal cells but not the retention or
organization of attracted lymphocytes. The related observations in
RIP-CXCL13 transgenic mice (9) suggest that several
chemokines can influence the development, growth, or activation of
stromal and endothelial cells by regulating LT12 expression on
lymphocytes. Consistent with this notion is the correlation between the
low numbers of stromal cells and addressin-expressing HEVs in
RIP-CXCL12 and RIP-CCL19 infiltrates and the relatively weak capacity
of these two chemokines to induce LT12 in vitro (Fig. 5, B–E).
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12 induction on T cells
Previous studies on RIP transgenic mice have shown that several
cytokines in addition to LT and TNF (41, 42), including
IL-2, IL-4, IL-6, IL-10, IFN-, and IFN-
, induce pancreatic
infiltrates (43). In addition, transgenic mice
overexpressing IL-7 develop lymphoid infiltrates in the skin (44, 45). To test the possibility that cytokines might promote
ectopic lymphoid tissue formation by inducing LT12 expression on
mature lymphocytes, we incubated splenocytes with various cytokines and
measured staining of LT12 with LTR-Fc. Strikingly, both IL-4
and IL-7 potently induced LT12 expression on all naive
(CD69-) CD4 and CD8 T cells (Fig. 7A) but not on B cells (data
not shown). Titration analysis of IL-4 and IL-7 showed that 0.2–20
ng/ml of each cytokine was sufficient to trigger LT12 induction
(Fig. 7B). IL-15 induced LT12 expression only on naive
CD8 T cells but not CD4 T cells (Fig. 7A). The induction of
LT12 expression by these cytokines occurred independently of
Gi-coupled receptor signals, as it could not be
blocked by pretreatment with Ptx (data not shown). A different pattern
was observed for spontaneously activated CD4 and CD8 T cells
(CD69+), as all IL-2 cytokine family members
(IL-2, -4, -7, -15) were able to further enhance already elevated
levels of surface LT12 (data not shown), consistent with the
up-regulation of the high-affinity receptor for IL-2 and IL-15 on
activated T cells. All other cytokines tested at concentrations of
2–20 ng/ml, including IL-6, IL-10, IL-12, IFN-, IFN-, IFN-,
and TNF, did not show any effect on LT12 induction in 6-h
cultures (data not shown). While these observations indicate that there
must be several mechanisms that lead to ectopic lymphoid cell
accumulation, they suggest that IL-4 and IL-7, and perhaps also IL-2,
promote lymphoid tissue formation by activating the LT12-LTR
pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12
up-regulation on naive CD4 T cells. Furthermore, two cytokines that
induce ectopic lymphoid tissue development, IL-4 and IL-7, were shown
to be active in up-regulating LT12 on naive CD4 and CD8 T cells.
A third IL-2 family cytokine, IL-15, induced LT12 on naive CD8 T
cells. These findings indicate that chemokine- and cytokine-mediated
induction of LT12 on naive T cells may participate in promoting
development of endogenous and disease-associated lymphoid tissues.
In two previous studies, CCL21 expression in the pancreatic islets was
observed to cause lymphoid neogenesis, with recruitment of lymphocytes
and DCs, and development of HEVs and stromal cells (22, 23). Our findings in three RIP-CCL21 transgenic lines confirm
the activity of CCL21 in promoting lymphoid neogenesis and extend these
findings by demonstrating that LT12 is required downstream of
CCL21 for development of HEVs and stromal cells. Surprisingly,
constitutive LT12 does not appear to be needed for the presence
of large numbers of lymphocytes in the islets, suggesting that
many of the cells are present due to the direct recruiting activity of
CCL21. It is also interesting that despite the greater chemotactic
activity of CCL19 and CCL21 for T cells than B cells, B cells often
dominate the pancreatic infiltrates. It remains to be determined
whether this effect is selective to the pancreas and whether factors in
addition to the chemokines are involved.
Coexpression of CCL21 and CCL19 in lymphoid tissues is a conserved
property between rodents and man, but the relative contribution of the
two chemokines to leukocyte recruitment is unclear. In recent studies
it was demonstrated that s.c. injection of CCL21 and CCL19 into
plt mice, which lack lymphoid CCL19 and CCL21 protein, led
to comparable T cell recruitment across HEVs in the draining lymph
nodes (21, 46). However, our findings revealed strikingly
lower amounts of CCL19 than CCL21 in lymph nodes and spleen of
wild-type mice despite similar mRNA levels. Our estimates for
CCL21 levels in lymph nodes and spleen, determined by Western blot, are
comparable to the previously reported values determined by ELISA
(22, 46). CCL19 protein levels have not been previously
reported. Based on our measurements, and assuming a tissue density of 1
g/ml, we estimate CCL21 concentrations in total spleen and lymph node
of 11,000–12,000 ng/ml and CCL19 concentrations of 20–40 ng/ml. As
the T cell areas in spleen and lymph nodes correspond to only
20–50% of the tissue area, the local chemokine concentrations
within the T zone are likely to be severalfold higher than these
estimates, with even higher concentrations at local points of
production and secretion. Importantly, both of these concentration
estimates are within ranges that have been found in vitro to induce
chemotaxis of T cells and DCs (for examples, see Refs. 18, 47 , and 48). Previous studies have shown that the
extended carboxyl-terminal domain of CCL21 contributes to increased
retention of this protein in lymphoid tissue, probably because its
highly basic properties favor interactions with extracellular
proteoglycans (46). The absence of this domain in
CCL19 may contribute to the poorer accumulation of the protein and
might in turn indicate that CCL21 and CCL19 differ substantially in the
extent and steepness of the chemoattractive gradients that they form.
Our findings suggest that CCL21 will play a more important role in
lymphoid tissue organization than CCL19.
Like CCL19 and CCL21, CXCL12 is a strong in vitro attractant of naive T cells (49). It is therefore significant that few T cells were recruited to the islets of RIP-CXCL12 transgenic mice. This may reflect insufficiency of CXCL12 for recruitment of T cells in vivo. The finding is also consistent with the possibility that CXCL12 has T cell chemorepellant activity (50). By contrast with the poor recruitment of T cells, CXCL12 caused notable accumulation of DCs, indicating that CXCL12 is sufficient to regulate the in vivo distribution of DCs. DCs are found near or within sites of endogenous CXCL12 expression, including the red pulp of the spleen (31) and in the gut and skin (32, 33, 51); therefore, we suggest that CXCR4 contributes to controlling DC positioning in both nonlymphoid and lymphoid tissues. The marked accumulation of plasma cells in RIP-CXCLl2 transgenic islets is in agreement with the recent finding that CXCL12 is important in directing plasma cell movements in vivo (31) and indicates that CXCL12 is sufficient to cause accumulation of these cells. This observation suggests that the CXCL12 expressed in the joint synovium of rheumatoid arthritis patients (34, 35) contributes to the marked local accumulation of plasma cells that is typical of this disease (52).
LT12 expression has been reported previously on activated but not
naive T cells (12, 53). We report low levels of LTR-Fc
binding to naive T cells in blood and lymphoid tissues that can be
blocked by an LT-specific Ab, suggesting low constitutive expression
of LT12 on T cells. The physiological significance of LT12
expression by recirculating T cells remains to be determined, but it
seems possible that it contributes to some aspects of lymphoid T zone
organization. Compared with the low expression on naive T cells ex
vivo, we find that CD4 T cells incubated in vitro with CCL21 or CCL19
express high levels of LT12. The minimal induction on CD8 vs CD4
T cells is unexpected because both cell types respond similarly to
CCL19 and CCL21 in chemotaxis assays and both up-regulate LT12 in
response to IL-4 and IL-7. These observations suggest that different
signaling pathways are activated by CCR7 within these two cell types.
Interestingly, the activity of CCL19 and CCL21 in promoting LT12
expression on CD4 T cells was similar to or greater than the activity
of CXCL13 in inducing expression on B cells (Fig. 5 and Ref.
8). In vivo, follicular CXCL13 up-regulates LT12 on
naive B cells entering secondary lymphoid organs (8).
However, we have not observed differential LT12 expression
between naive T cells from blood vs spleen and lymph nodes. These
observations appear paradoxical in the light of the 50- to 250-fold
higher expression of CCL21 compared with CXCL13 within secondary
lymphoid tissues, and suggest that LT12 expression on naive CD4 T
cells is negatively regulated within lymphoid tissue. It remains to be
determined whether this is also the case at sites of inflammation.
Comparing the in vitro activities of CCL21 and CCL19 in LT12
induction on CD4 T cells revealed that CCL21 was more effective than
CCL19. In addition to the many studies that have shown LT12 is
necessary for lymph node and Peyer’s patch development
(10), LT expression has been shown to be sufficient to
mediate lymphoid neogenesis in islets of transgenic mice
(42). More recently, we demonstrated that development of
organized infiltrates in response to ectopic CXCL13 was dependent on
LT12 (9). Therefore, the larger infiltrates induced
by CCL21 compared with CCL19 may reflect the stronger induction of
LT12 by CCL21, in addition to the larger amounts of this
chemokine present in the pancreas. CCL21-induced infiltrates have
previously been shown to develop in the absence of B cells but not in
the combined absence of T and B cells (22), supporting a
role for T cell-derived LT12 in promoting downstream effects in
the infiltrate. Therefore, we propose that naive CD4 T cells attracted
to the CCL21-expressing islets are induced to up-regulate LT12,
leading to the development of HEVs and lymphoid stromal cells. Previous
observations have shown an important role for LT12 in the
induction of CXCL13 and CCL21 and, to a lesser extent, CCL19 in
secondary lymphoid tissues (54). The subsequent finding
that CXCL13 could induce LT12 on B cells led to the discovery of
a positive feedback loop between LT12 and CXCL13
(8). Although we have not been able to determine whether
endogenous CCL21 is induced downstream of ectopically expressed CCL21,
our findings are consistent with the possibility that CCL21 and
LT12 act together in a positive feedback loop. The possible
pathological relevance of the relationship between CCL21 and LT12
is supported by the finding of CCL21 mRNA (26) and protein
(Fig. 2D) in the pancreatic infiltrates of NOD mice and the
recent evidence that LT12 is critical for development of diabetes
in NOD mice (55, 56). It will be interesting in future
studies to test whether blocking CCL21 function is sufficient to
prevent development of diabetes in NOD mice.
The findings that chemokine-mediated ectopic lymphoid tissue formation
correlated with the activity of chemokines in inducing LT12
expression led us to ask whether cytokines that promote lymphoid
infiltrates also induce LT12 on naive lymphocytes. This inquiry
revealed that IL-4, IL-7, and IL-15 were potent in up-regulating
LT12 expression in vitro on naive T cells, and all IL-2 cytokine
family members promoted up-regulation on activated T cells. In this
regard, it is significant that IL-4-induced infiltrates have been shown
to contain MAdCAM-positive HEVs and stromal cells (43).
Consistent with our observations, IL-2 has been shown to induce LT
mRNA in spleen and lymph node cells (57). In other
studies, LT has been characterized as a Th1 cytokine, and Gramaglia
et al. (12) observed that Th2 cells do not express
LT12, and combined IL-4 and anti-IFN- treatment inhibited
expression on activated T cells. We suspect that the differences
between this latter study and our findings of LT12 induction by
IL-4 reflect that we treated freshly isolates splenocytes, whereas
Gramaglia et al. (12) examined effects on TCR-activated
cells. In other studies it has been shown that IL-7 up-regulates
LT12 on
CD4+CD3-IL7R+
Peyer’s patch-inducing cells (58). IL-7 is constitutively
expressed within secondary lymphoid organs and is critical for survival
of naive T cells (59, 60, 61). It is tempting to speculate
that LT12 induction on T cells by IL-7 is part of the cross-talk
occurring between recirculating T cells and LTR- and IL-7-expressing
T zone stromal cells. A general role of cytokine-mediated induction of
LT12 on naive lymphocytes may be to allow bystander cells to help
reorganize tissue within the lymphoid organ or at a site of
inflammation to better support the proliferation of the responding
Ag-specific cells.
Finally, several recent studies have investigated the effect of lymphoid tissue chemokines on the antitumor immune response in mice. Introduction of CCL21 into the tumor caused increased infiltration by DCs, T cells, and in some cases granulocytes, and was associated with tumor regression (62, 63, 64). A comparison of the effects of transducing tumors with CCL19, CCL21, or CXCL12 in promoting tumor regression demonstrated that CCL21 was most effective, CXCL12 was intermediate, and CCL19 was least effective (65). Tumor targeting of LT was also recently shown to enhance T cell-mediated tumor regression (3). These combined observations agree well with our findings that CCL21 is highly effective in promoting T cell recruitment, LT up-regulation, and tissue invasion, and support further testing of this chemokine in antitumor immunotherapy.
|
Acknowledgments |
|---|
R-Fc and Ab
(BBF6), and Drs. Jeff Browning and Richard Locksley for critical
comments on the manuscript. |
Footnotes |
|---|
2 A.B. and D.C.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jason G. Cyster, Department of Microbiology and Immunology, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0414. E-mail address: cyster{at}itsa.ucsf.edu
4 Abbreviations used in this paper: HEV, high endothelial venule; NOD, nonobese diabetic; DC, dendritic cell; LT, lymphotoxin; Ptx, pertussis toxin; PNAd, peripheral lymph node addressin; MAdCAM, mucosal addressin cell adhesion molecule; RIP, rat insulin promoter; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication February 20, 2002. Accepted for publication April 16, 2002.
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H. Dalwadi, B. Wei, M. Schrage, T. T. Su, D. J. Rawlings, and J. Braun B Cell Developmental Requirement for the G{alpha}i2 Gene J. Immunol., February 15, 2003; 170(4): 1707 - 1715. [Abstract] [Full Text] [PDF]
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J. L. Pablos, B. Santiago, M. Galindo, C. Torres, M. T. Brehmer, F. J. Blanco, and F. J. Garcia-Lazaro Synoviocyte-Derived CXCL12 Is Displayed on Endothelium and Induces Angiogenesis in Rheumatoid Arthritis J. Immunol., February 15, 2003; 170(4): 2147 - 2152. [Abstract] [Full Text] [PDF]
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, Upper limits of the amount of
chemokine that could have been present in samples where no signal was
detected. E and F, Real-time quantitative
RT-PCR analysis of CCL19 and CCL21 mRNA levels relative to HPRT mRNA
level in samples from transgenic pancreas (E) or
nontransgenic (wild-type) lymph node and spleen (F). A
titration analysis with CCL19 and CCL21 plasmid DNA established a
3-fold lower amplification efficiency for CCL19 vs CCL21, and the
threshold cycle values obtained for the CCL19 mRNA samples were
multiplied by a factor of three to correct for this difference in
reaction efficiency. Shown in E are relative chemokine
expression levels in nontransgenic (tg-) pancreas as well as in
RIP-CCL-19 transgenic pancreas from mouse lines I and II and in
RIP-CCL21 transgenic pancreas from mouse lines II and IV.
