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* First Department of Internal Medicine and
Department of Pathology, Ehime University School of Medicine, Ehime, Japan
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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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is thought to play a prominent role (8, 9). In contrast, chronic GVHD has a more indolent course, and
has more diverse pathologic manifestations. The clinical presentation
can resemble that of systemic lupus erythematosus, and is characterized
by the development of immune complex disease and autoantibody
formation. Although this form of GVHD is thought to be mediated by a
Th2 type humoral immune response, the mechanism of chronic GVHD is not
fully understood. Murine chronic GVHD resembling human chronic GVHD induced by PBSCT can be induced by the transfer of DBA/2 (H-2d) mouse spleen cells into (C57BL/6 x DBA/2) F1 (B6D2F1) (H-2b/d) mice. In this model, mice exhibit a systemic autoimmune disorder characterized by splenomegaly, host B cell hyperactivation, autoantibody production, and immune complex deposition (10, 11). In addition, mice with chronic GVHD exhibit increases in the number of total spleen cells, including host B cells, and in the levels of serum IgE, and of total IgG and IgG1 of anti-DNA Abs after chronic GVHD induction (12). It has been postulated that alloreactive donor (DBA/2) CD4+ T cells recognize MHC class II molecules on the B6D2F1 host cells, and then provide help to host B cells for autoantibody production (10, 13).
Chemokines are often chemotactic and mitogenic for specific immune
cells. Several new lymphocyte-specific chemokines, which attract naive
and memory T cells, B cells, NK cells, and dendritic cells, have been
reported (14, 15). Secondary lymphoid- tissue chemokine
(SLC)/6Ckine/CCR ligand 21 and EBI-1-ligand chemokine
(ELC)/macrophage inflammatory protein (MIP)-3
/CCR ligand 19 are
strongly expressed in T cell zones of lymphoid organs, and SLC has been
localized to high endothelial venules (HEVs) and lymphatic endothelium,
suggesting that SLC and ELC play important roles in the homing of
lymphocytes and dendritic cells to lymphoid tissues
(16, 17, 18, 19, 20, 21, 22). The chemokine receptor CCR7, the functional
receptor for SLC and ELC, is expressed in CD4+
and CD8+ T cells as well as in B cells, but not
in NK cells, monocytes, or neutrophils in peripheral blood. CCR7
expression is up-regulated in B cells infected by EBV,
CD4+ T cells infected by human herpesvirus-6 or
human herpesvirus-7, mature dendritic cells, and adult T cell leukemia
cells (23, 24, 25). plt mice, which lack SLC and
have reduced ELC production, have been reported to have a phenotype
resembling that of CCR7-deficient mice (26, 27). Both
mutant mice show marked disturbances in the distribution of T, B, and
dendritic cells, resulting in impaired T cell priming and T
cell-dependent B cell response. Therefore, SLC and ELC have potent
chemotactic activity for CD4+ and
CD8+ T cells, B cells, and mature dendritic cells
and potentially play a role in T cell priming by colocalization of
Ag-presenting dendritic cells and T cells.
The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. In several reports, N-terminally truncated or modified chemokine analogs have been described to act as receptor antagonists (28, 29, 30, 31, 32, 33, 34, 35, 36). We describe the receptor antagonist properties of murine N-terminally truncated SLC analogs and evaluate the preventive effects of SLC antagonist treatment on chronic GVHD model mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CCR7-transfected L1.2 cells (L1.2/CCR7) were kindly provided by O. Yoshie (Kinki University School of Medicine, Osaka, Japan). This transfectant was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD). DBA/2 (H-2d) and (C57BL/6 x DBA/2)F1 (B6D2F1) female mice, 6–8 wk of age, were purchased from Charles River Breeding Laboratories (Atsugi, Kanagawa, Japan) and housed for 2 wk before GVHD induction.
Abs and reagents
FITC-conjugated anti-mouse I-Ab
(Ab) (AF6-120.1) and FITC-anti-mouse
H-2Kb (AF6-88.5) mAbs were purchased from BD
PharMingen (San Diego, CA). PE-conjugated anti-mouse CD4 (KT6),
PE-anti-mouse CD8 (KT15), and PE-anti-mouse B220 (RA3.6B2) mAbs
were obtained from Immunotech (Marseilles, France). Recombinant mouse
SLC (6Ckine) was purchased from R&D Systems (Minneapolis, MN).
Construction of expression vectors encoding functional murine SLC chemokine and their N-terminal truncated analogs
To amplify the fragments containing functional coding regions, the following specific primers of murine SLC were used: 5'-AGTGATGGAGGGGGTCAGGAC-3' (forward) and 5'-CAGGCGGGCTACTGGGCTATC-3' (reverse) (37). Forward primers for each N-terminal truncated SLC analog were prepared with 21 oligonucleotide sequences corresponding to each truncated analog.
Total cellular RNAs were extracted from spleens of DBA/2 mice as described previously (38). The above fragments were amplified by RT-PCR by using an RNA PCR kit (Takara Shuzo, Kyoto, Japan) as described previously (39). After confirming the entire nucleotide sequence, the fragment was cloned into the KpnI site of pThioHis A vector (Invitrogen, Carlsbad, CA) after this site was end-filled to give a blunt end.
Purification of functional murine SLC chemokine protein and their analogs
The functional murine SLC chemokine protein and their analogs were purified as described previously (40). The in-frame pThioHis A-chemokine (SLC or their analogs) was transfected into Escherichia coli, TOP 10 strain, and His-patch-thioredoxin fusion proteins were prepared by using the His-Patch ThioFusion Expression system (Invitrogen) and purified on a ProBond resin column following the manufacturer’s instructions. After the fusion proteins had been digested with Enterokinase Max (Invitrogen), the complete chemokine proteins were purified by reverse-phase HPLC (RESOURCE RPC; Amersham Pharmacia Biotech, Piscataway, NJ). The protein concentration was determined with a BCA kit (Pierce, Rockford, IL), and the purity was analyzed by SDS-PAGE and silver staining. The purity of chemokine proteins was >95%.
Chemotactic assay
Chemotactic assays for spleen cells were performed in polycarbonate-membrane, 6.5-mm diameter, 5-µm pore size transwell cell culture chambers (Costar, Cambridge, MA), as described previously (25). Aliquots (100 µl) of cells (5 x 106/ml) suspended in RPMI 1640/0.5% BSA were added to the upper chambers. Either SLC or analog, to produce a final concentration of 5 µg/ml, was added to the lower wells. The cells were allowed to migrate for 2 h at 37°C in a 5% CO2 incubator, after which the filters were fixed with 1% glutaraldehyde in PBS for 30 min and stained with 0.5% toluidine blue overnight. Cell migration was quantified by counting cells in each lower chamber and cells adhering to the bottom part of the polycarbonate filter. For blocking assays, after reacting with each analog at a final concentration of 1 or 5 µg/ml at 37°C for 30 min, the cells were added to the upper chambers, and chemokine was added to the lower wells at a final concentration of 5 µg/ml. Each assay was performed in triplicate.
Analysis of cytosolic-free calcium
L1.2/CCR7 cells (4 x 105) were loaded with 12.5 mg/ml Fluo-3AM (Nacalai Tesque, Kyoto, Japan) in PBS with 0.38 mg/ml Pluronic F127 (Molecular Probes, Eugene, OR) at 37°C for 30 min. After washing with PBS, the cells were resuspended in 25 mM HEPES, 140 mM NaCl, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2, and 3 mM KCl, pH 7.3. The fluorescence was monitored at 7-s intervals over 150 s, after addition of the test sample. Maximum Ca2+ levels were established using Fluo-3AM (designated 100% saturation) for each set of measurements by addition of 5 mM ionomycin (Sigma-Aldrich, St. Louis, MO). Ca2+ desensitization was performed by addition of one ligand, and then after 150 s the cells were treated with either the same or a different ligand.
Receptor binding
Receptor binding of analogs was assayed by competition with 125I-labeled SLC. First, murine SLC (10 µg) was labeled with monoiodinated Bolton-Hunter reagent (specific activity 2200 Ci/mmol; DuPont, Wilmington, DE) at 4°C for 30 min. To determine the binding kinetics, L1.2/CCR7 cells (5 x 106) in 200 µl of binding buffer (RPMI 1640 medium, 0.5 mg/ml BSA, 50 mM HEPES, and 0.1% NaN3) were incubated with 125I-labeled SLC and varying concentrations of unlabeled competitor (analog) at 4°C for 30 min. The cells were pelleted through a mixture of diacetylphthalate and dibutylphthalate, and the cell-associated radioactivity was counted (total binding). Nonspecific binding was determined in the presence of a 100-fold concentration of unlabeled ligand and was subtracted from the total binding. Dissociation constants (Kd values) were determined by Scatchard analysis.
Preparation of murine cells and induction of chronic GVHD
Single-cell suspensions were prepared in RPMI 1640 from the spleens of normal DBA/2 parental donors. Cell suspensions were filtered through sterile mesh and washed. After the erythrocytes had been lysed in hemolysis buffer (including 144 mM NH4Cl and 17 mM Tris-HCl, pH 7.2), DBA/2 spleen cells were suspended at 90 x 106 viable cells/0.25 ml in RPMI 1640 medium. Chronic GVHD was induced by the injection of 90 x 106 DBA/2 (H-2d) mice parental cells into the tail vein of a normal, unirradiated B6D2F1 (H-2b/d) mouse. In the control PBS-treated and the SLC antagonist-treated groups, 90 x 106 DBA/2 spleen cells per mouse were injected into B6D2F1 recipients after incubation with PBS and murine SLC antagonist at a concentration of 50 µg/ml in RPMI 1640 medium supplemented with 2% FCS, at room temperature for 1 h, rotating with ROTATOR RT-50 (TAITEC, Saitama, Japan), respectively.
To determine whether in vivo administration of SLC antagonist inhibits the development of chronic GVHD, we designed the following administration groups: group 1, i.v. injection of 30 µg/mouse four times, 2 h before, just before, 2 h after, and 6 h after GVHD induction; and group 2, i.v. injection of 60 µg/mouse twice, 2 and 6 h after GVHD induction. These mice were observed for 16 days after GVHD induction.
Flow cytometric analysis of parental cell engraftment
The spleens of GVHD mice were harvested at 1, 5, 8, and 16 days after inoculation. Single-cell suspensions of splenocytes were prepared, and the number of cells per spleen was counted. Flow cytometric analysis was performed as described previously (41). After the erythrocytes had been removed with hemolysis buffer, the splenocytes were stained with FITC-conjugated anti-H-2b to distinguish parental from B6D2F1 cells. To further identify the splenocyte populations, anti-CD4, anti-CD8, and anti-B220 conjugated to PE were used. Host-B cell MHC class II expression is shown as the mean channel fluorescence for host Ia on B220+ lymphocytes using FITC-conjugated anti-I-Ab. Donor T cell engraftment is shown as the number of CD4+ or CD8+ lymphocytes that did not stain positively for MHC class II of the opposite parent. Data were collected on a FACScan flow cytometer and analyzed with CellQuest software (BD Biosciences, San Jose, CA).
Immunohistochemistry
For immunohistochemical analysis, cells were labeled with biotin sulfo-NHS-LC-biotin (Pierce) at a final concentration of 1 mg/ml in PBS with 5% FCS at 37°C for 30 min. After washing with PBS, cells (90 x 106/mouse) were suspended in RPMI 1640/2% FCS with or without murine SLC antagonist and injected into the tail veins of B6D2F1 mice. After 5 h, the mice were sacrificed, and their spleens were embedded in OCT (Tissue Tek, Elkhart, IN), snap-frozen on dry ice, and then stored at -70°C. Cryostat sections (5-µm thick) on slides were dried overnight and washed three times in PBS. The sections were incubated at room temperature for 15 min in 3% H2O2 to inhibit endogenous peroxidase activity, and then incubated with streptavidin/HRP (DAKO, Carpinteria, CA) at room temperature for 1 h. They were developed with a 3-amino-9-ethylcarbazole (AEC) substrate chromogen system (AEC as chromogen; DAKO). The sections were counterstained with hematoxylin.
Autoantibody detection
Serum titers of total IgG and IgG1 of anti-DNA Abs were assessed by ELISA. The 96-well Maxisorp plates (Nalge Nunc International, Roskilde, Denmark) were incubated overnight at 4°C with 50 µl of calf thymus DNA (Sigma-Aldrich) at a concentration of 5 µg/ml in PBS. After blocking with 2% BSA, plates were incubated with 50 µl of diluted serum samples for 2 h at room temperature. After washing three times with PBS containing 0.1% Tween 20, HRP-labeled goat anti-mouse IgG or IgG1 was added to each well and the plates were kept at room temperature for 2 h. After the plates had been washed with PBS containing 0.1% Tween 20, the color was developed for 15 min in 100 µl of 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich), stopped by adding 100 µl of 1 N HCl. The plates were read at 450 nm with IMMUNO-MINI (Nalge Nunc International) within 30 min.
Quantification of murine IL-2, IFN-, and IL-4
Spleen cells (5 x 106) were cultured
in 1 ml of RPMI 1640 medium supplemented with 10% FCS, 5 mM 2-ME, and
10 µg/ml Con A (Sigma-Aldrich). The culture supernatants were
harvested after 24 and 48 h and analyzed for murine IL-2, IFN-,
and IL-4 with ELISA kits (Endogen, Webura, MA). Each sample was assayed
in triplicate and the mean values were calculated.
Statistical analysis
Statistical analysis was performed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the role of the N-terminal region of murine SLC in
determining function, we designed a
series of analogs that were shortened at the N terminus (Fig. 1
). As
shown in Fig. 2, at a concentration of 5
µg/ml, three N-terminally truncated analogs, murine (m)SLC-1,
mSLC-2, and mSLC-3, had lower chemotactic activity for L1.2/CCR7 cells
(58 ± 11%, 55 ± 12%, and 42 ± 8%, respectively;
p < 0.01) than that of the complete form of murine
SLC. Another three analogs, mSLC-4, mSLC-5, and mSLC-6, which were
truncated >4 aa residues from the N terminus, failed to induce
chemotactic activity. Similar results were obtained with mouse spleen
cells as the target cells (data not shown).
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). Induction of cytosolic
Ca2+ mobilization by the N-terminally truncated
analogs generally correlated with the results of chemotaxis. The
mSLC-1, mSLC-2, and mSLC-3 induced a lower response (42 ± 11%,
38 ± 8%, and 30 ± 14%, respectively; p <
0.01) than that of the complete form, whereas mSLC-4, mSLC-5, and
mSLC-6 did not induce significant cytosolic Ca2+
at a concentration of up to 50 µg/ml. From these findings, we suggest
that those analogs truncated >4 aa residues from the N terminus showed
loss of ability to induce chemotaxis and Ca2+
influx. Blocking of chemotaxis and Ca2+ influx by murine N-terminally truncated SLC analogs
Next, we examined whether the three analogs, mSLC-4, mSLC-5, and
mSLC-6, worked as antagonists. The mSLC-4, mSLC-5, and mSLC-6 inhibited
SLC-stimulated chemotaxis (95 ± 5%, 90 ± 5%, and 92
± 7% reduction, respectively; p < 0.01) and
SLC-induced Ca2+ influx (97 ± 3%, 93
± 5%, and 97 ± 3% reduction, respectively; p
< 0.01) at a concentration of 5 µg/ml (Fig. 3).
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SLC antagonist prevents the development of chronic GVHD
To determine whether SLC antagonist inhibits the development of
chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells
into B6D2F1 mice. In a preliminary experiment, we
examined the optimum concentration of mSLC-4 for blocking the
chemotaxis of DBA/2 spleen cells using in vitro chemotactic assays. At
a concentration of >20 µg/ml for 90 x
106 DBA/2 spleen cells, the decrease of
chemotaxis reached a plateau (data not shown). Therefore, we used
mSLC-4 at a concentration of 50 µg/ml for this experiment.
Furthermore, we compared cell viability, proliferation in response to
the T cell mitogen Con A, and cytokine production between PBS-treated
and SLC antagonist-treated splenocytes 5 h after treatment. There
were no differences in cell viability (>90%) or Con A-stimulated
proliferation between these two groups (data not shown). In addition,
we also confirmed that there was no difference in production of
cytokines such as IL-2, IL-4, and IFN- after Con A stimulation
between these two groups (data not shown). After incubation with
mSLC-4, spleen cells were injected into the tail veins of
B6D2F1 mice. On day 16 after the induction,
control PBS-treated mice exhibited the following findings typical of
chronic GVHD, as described previously (10): splenomegaly,
an increase in numbers of host B cells, and elevated serum levels of
IgE and of IgG and IgG1 of anti-DNA Abs. The total number of spleen
cells on day 16 after GVHD induction increased 1.8-fold: the number of
cells on day 16 after GVHD induction was (105 ± 12) x
106 vs before GVHD induction (58 ± 8)
x 106 (p < 0.05) (Fig. 4). The number of host B cells increased
2.2-fold: the number of cells on day 16 after GVHD induction was
(62 ± 10) x 106 vs before GVHD
induction (28 ± 6) x 106
(p < 0.05). In contrast, host
CD4+ and CD8+ T cells
possessed only 9.0% ((5.2 ± 0.8) x
106) and 7.0% ((4.0 ± 0.5) x
106) of total spleen cells, respectively, and
their numbers had increased 1.4-fold ((7.3 ± 1.2) x
106 (p < 0.05)) and
1.2-fold ((4.6 ± 0.5) x 106
(p < 0.05)) on day 16 after GVHD induction.
Therefore, the increase in numbers of spleen cells after GVHD induction
is mainly due to the increase in numbers of host B cells. GVHD mice
treated with SLC antagonist exhibited signs of chronic GVHD, but these
signs were reduced compared with those of control mice. On day 16 after
induction, total numbers of spleen cells and host B cells in SLC
antagonist-treated GVHD mice were reduced to 67% ((70 ± 8)
x 106 (p < 0.05)) and
40% ((25 ± 8) x 106
(p < 0.05)), respectively, of the values in the
control PBS-treated GVHD mice, whereas there was no significant
difference in the numbers of either CD4+
((6.3 ± 0.7) x 106 vs (7.3 ±
1.2) x 106) or CD8+
((4.4 ± 0.3) x 106 vs (4.6 ±
0.5) x 106) host T cells between SLC
antagonist-treated and control PBS-treated GVHD mice.
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). In addition, the serum
IgG1 of anti-DNA Abs and IgE levels in SLC antagonist-treated GVHD
mice were also reduced to 31% (OD: 0.041 ± 0.020 vs 0.132
± 0.032 (p < 0.05)) and 52% (1.15 ±
0.20 vs 2.22 ± 0.28 µg/ml (p < 0.05))
of those in control PBS-treated GVHD mice, respectively. These findings
showed that SLC antagonist ameliorated chronic GVHD. Furthermore, we
confirmed these findings by analyzing survival 16 days after
transplantation. As shown in Fig. 6, SLC-antagonist-treated GVHD mice showed a significant increase in
survival compared with control PBS-treated GVHD mice (median survival:
>80 vs 52 days, respectively; p < 0.05).
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, the control PBS-treated
donor cells were spread diffusely within the periarteriolar lymphoid
sheaths (PALS). In contrast, the number of SLC antagonist-treated donor
cells in the PALS were reduced markedly (by 83%, p <
0.01). These findings suggest that addition of SLC antagonist inhibited
the development of chronic GVHD by reducing the infiltration of
CCR7-positive donor lymphocytes into the PALS of the recipient’s
spleen.
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To examine the mechanism by which the SLC antagonist prevents the
development of chronic GVHD, we compared subsets of donor lymphocytes
in the spleen between control PBS-treated GVHD mice and SLC
antagonist-treated GVHD mice. Donor CD4+ T cells
in control PBS-treated GVHD mice were activated and were increased in
number, 9-fold ((3.5 ± 0.8) x 106
(p < 0.05)) on day 5 after GVHD induction,
11-fold ((4.4 ± 0.6) x 106
(p < 0.05)) on day 8, and 8-fold ((3.3 ±
1.1) x 106 (p <
0.05)) on day 16, compared with those on day 1 after GVHD induction
((0.4 ± 0.2) x 106) (Fig. 8). In contrast, donor
CD8+ T cells in PBS-treated GVHD mice were not
activated as efficiently and increased in number <2-fold ((1.2 ±
0.8) x 106, (1.6 ± 0.7) x
106, and (0.8 ± 0.4) x
106 on days 5, 8, and 16 after GVHD induction,
respectively), compared with those on day 1 after GVHD induction
((0.8 ± 0.6) x 106). The numbers of
donor CD4+ T cells in the spleens of SLC
antagonist-treated GVHD mice were reduced to 51% ((1.8 ±
0.8) x 106), 50% ((2.2 ± 1.0)
x 106), and 57% ((1.9 ± 1.0) x
106) on days 5, 8, and 16, respectively
(p < 0.05), of the values in control
PBS-treated GVHD mice. In contrast, over the 16 days after GVHD
induction, there were no significant differences in the numbers of
donor CD8+ T cells in the spleen between SLC
antagonist-treated GVHD mice and control PBS-treated GVHD
mice.
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and IL-2 production between SLC antagonist-treated GVHD mice
and control PBS-treated GVHD mice (Table I). From these findings, the amelioration
of chronic GVHD by treatment with an SLC antagonist appears to be due
to a reduction in the numbers of activated donor
CD4+ T cells and a decrease in IL-4 production,
resulting in a reduction in the numbers of activated host B cells.
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We examined whether in vivo administration of SLC antagonist
prevented the development of chronic GVHD. For this experiment, we
designed the following two different administration groups: group 1,
i.v. injection of 30 µg/mouse four times, 2 h before, just
before, 2 h after, and 6 h after GVHD induction; and group 2,
i.v. injection of 60 µg/mouse twice, 2 and 6 h after GVHD
induction. As shown in Fig. 9A, total numbers of spleen
cells and host B cells in group 1 were reduced to 71 and 67%,
respectively (p < 0.05), of the values in the
control PBS-treated GVHD mice on day 16 after induction, whereas there
were no significant differences in the numbers of spleen cells and host
B cells between group 2 and control PBS-treated GVHD mice. Amelioration
of chronic GVHD in group 1 was also supported by findings such as a
reduction in the numbers of donor CD4+ T cells in
the recipient’s spleen (Fig. 9B) and a decrease in the
serum levels of IgE, and of IgG and IgG1 of anti-DNA Abs (Fig. 9C). These results indicate that SLC antagonist administered
after transplantation cannot prevent the development of chronic GVHD
sufficiently, since donor T cells enter the lymphoid organs early after
injection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Truncation or modification of the few N-terminal amino acids of
chemokines has been reported to lead to significant changes in
functional activity and receptor binding (28, 29, 30, 31, 32, 33, 34, 35, 36).
Deletion of the pyroglutamate residue at the N terminus of the complete
form of monocyte chemoattractant protein (MCP)-1 results in at
least a 50-fold decrease of agonistic activity on monocytes and
basophils (28, 29). Deletion of the first two amino acids
results in almost total loss of activity, although surprisingly,
activity is regained on deletion of further amino acids. In the case of
IL-8, the N-terminal residues 4, 5, and 6 are essential for receptor
binding and triggering function (35, 36). RANTES loses
agonistic potency and becomes a potent antagonist of chemokine binding
when the first amino acid residue is modified artificially by addition
of methionine or treatment with aminooxypentane (30, 32, 33). The naturally cleaved forms of MCP-2 and RANTES are also
devoid of bioactivity (31, 34). The function of chemokines
has also been further clarified from crystal structure determination
and nuclear magnetic resonance data (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56). In
several chemokines such as IL-8, MCP-1, MCP-2, MCP-3, eotaxin, RANTES,
MIP-1
, MIP-1, fractalkine, and stromal cell-derived
factor-1, it has been observed that the N-terminal region is
essential for functional activity and that the loop immediately
following the first two cysteines in the sequence, as well as the
N-terminal region, plays an important role in receptor binding. We
found that the N-terminally truncated murine SLC had lower activity and
receptor binding than the complete form of murine SLC. The three
analogs, mSLC-1, mSLC-2, and mSLC-3, which were truncated up to three
amino acids from the N terminus, had only a 2-fold lower potency than
the complete form of murine SLC. With further deletions from the mSLC-3
analog, biological activity was lost but receptor binding was retained
with mSLC-4, mSLC-5, and mSLC-6. These findings show that at least four
amino acid residues preceding the first cysteine are essential for the
biological activity of murine SLC. Human SLC gives similar results to
murine SLC (our unpublished observations).
Chronic GVHD remains the most common complication of allogeneic stem cell transplantation (SCT). This is probably related to changes in clinical SCT practice. The use of unrelated donors and related but nonhuman leukocyte Ag (non-HLA)-identical donors is expanding. The use of donor lymphocyte infusion to treat relapsed disease or to achieve full donor chimerism has resulted in the development of chronic GVHD. Furthermore, there is evidence to suggest that patients receiving allogeneic PBSCT have a lower incidence of acute GVHD but an equally high or a higher incidence of chronic GVHD than comparable patients receiving marrow grafts (3, 4, 5, 6, 7). Although the reason for this is unknown, it may be due to the transfer of a significantly larger dose of mature, immunocompetent T cells. Until now, two mechanisms have been suggested for the pathogenesis of chronic GVHD (1, 2). One is alloreactivity to minor histocompatibility Ags, and the other is a role of postthymic CD4+ T cells. The T cell precursors may undergo aberrant "thymic education" after SCT—a process that effectively makes them autoreactive. However, the exact pathogenesis of chronic GVHD remains ambiguous. Murine chronic GVHD resembling human chronic GVHD induced by PBSCT can be induced by the transfer of DBA/2 (H-2d) mouse spleen cells into (C57BL/6 x DBA/2) F1 (B6D2F1) (H-2b/d) mice (10). This model is a chronic disorder characterized by a process in which donor CD4+ T cells become activated, secrete predominantly Th2-associated cytokines, and stimulate autoreactive B cells to differentiate into autoantibody-secreting cells. Serum Ig levels, including IgE levels, are elevated in these mice, whereas the function of in vitro CD8+ CTL is reduced and donor anti-host CTL are not detected. An autoimmune disease resembling human systemic lupus erythematosus also develops in this model.
It has been reported that CCR7 and its ligands, SLC and ELC, play a central role in the homing and traffic of T cells and dendritic cells into secondary lymphoid organs such as the peripheral lymph nodes, spleen, and Peyer’s patches (14, 15, 16, 17, 18, 19, 20, 21, 22). Lymphocyte transmigration from peripheral blood to secondary lymphoid organs occurs through HEVs and is a multistep process involving selectin-supported rolling, followed by a triggering event, and then firm integrin-mediated adhesion (57). SLC is highly expressed by HEVs and is active in inducing integrin-mediated adhesion of lymphocytes (18, 20). Once lymphocytes have crossed HEVs in lymph nodes and Peyer’s patches, T cells localize efficiently in T cell-rich areas, and B cells in B cell-rich areas. SLC, in addition to being expressed by HEVs, is expressed by stromal cells within T cell-rich areas, while ELC is produced by macrophages, by dendritic cells, and by some nonhemopoietic cells in T cell-rich areas (19, 20, 21, 22). In both CCR7-deficient mice and plt mice, the organization of cells in T cell-rich areas is severely disturbed, and there is also defective entry of cells across HEVs (26, 27). Therefore, CCR7 and its ligands are not only required for guiding T cells through HEVs, but also for directing T cells to their corresponding functional microenvironment once they have entered the lymphoid organs. On the basis of these results, we aimed at ameliorating chronic GVHD using an SLC antagonist that blocked the homing of donor CCR7-positive T cells into secondary lymphoid organs in which donor CD4+ T cell-host B cell interaction is mainly elicited. Infiltration into the PALS of the recipient’s spleen of CCR7-positive donor lymphocytes incubated with SLC antagonist was markedly reduced, compared with infiltration in control GVHD mice. Consequently, the number of activated donor CD4+ T cells and IL-4 production in SLC antagonist-treated GVHD mice were reduced significantly, resulting in a decrease in the numbers of activated autoreactive host B cells. This was also proven from the finding that serum levels of IgE, and of IgG and IgG1 of anti-DNA Abs, in SLC antagonist-treated GVHD mice were significantly lower than those in control GVHD mice.
Several studies have reported the role of chemokines and their
receptors in the pathogenesis of GVHD (58, 59, 60). In
addition to production of MIP-1 by donor T cells, MIP-1
expression is significantly increased in acute GVHD target organs such
as the liver, lung, and spleen after transfer of allogeneic lymphocytes
compared with syngeneic lymphocytes (58, 59). Treatment
with anti-MIP-1 or anti-CCR5 Abs reduces liver damage in
GVHD (58). These results suggest that MIP-1-induced
migration of CCR5-expressing CD8+ T cells plays a
significant role in the occurrence of acute GVHD. In a murine
sclerodermatous GVHD model, early elevated cutaneous mRNA expression of
TGF-1, elevated CC chemokines, MCP-1, MIP-1, and RANTES preceded
subsequent skin and lung fibrosis, suggesting that TGF-1-producing
donor mononuclear cells and these chemokines might be important in the
early pathogenesis of sclerodermatous GVHD (60).
Therefore, these findings and our present results indicate that
receptor antagonists for chemokines could be considered as therapeutic
target for the prevention and treatment of GVHD.
Finally, there are several problems in applying the findings obtained from this model to the treatment of GVHD in humans. T cell depletion of the donor graft can dramatically reduce the incidence of GVHD (reviewed in Refs. 1 and 2). However, this reduction in GVHD did not translate into improved overall survival because of delayed immune reconstitution and unexpectedly high rates of graft failure and disease recurrence. Because SLC antagonist-treated donor T cells cannot traffic sufficiently to areas that contain donor-reactive host T cells, treatment with SLC antagonist may affect immune reconstitution and graft failure. In human allogeneic transplantation, recipients are treated with a cytotoxic regimen with additional whole-body irradiation to reduce the frequency of graft rejection. However, this treatment is associated with increased frequency and severity of GVHD with histopathological damage. In contrast, the model in this study is a chronic GVHD model with little to no histopathological damage, in which unirradiated B6D2F1 mice are infused with DBA/2 spleen cells. Therefore, although the mechanism of chronic GVHD induced in this mouse model is not necessarily identical with that of chronic GVHD in humans, SLC antagonist may provide a new potentially useful approach for the prevention of chronic GVHD.
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2 Address correspondence and reprint requests to Dr. Hitoshi Hasegawa, First Department of Internal Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan. E-mail address: hitoshih{at}m.ehime-u.ac.jp
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; SCT, stem cell transplantation; PBSCT, peripheral blood SCT; SLC, secondary lymphoid tissue chemokine; ELC, EBI-1-ligand chemokine; HEV, high endothelial venule; MIP, macrophage inflammatory protein; AEC, 3-amino-9-ethylcarbazole; m, murine; PALS, periarteriolar lymphoid sheath; MCP, monocyte chemoattractant protein.
Received for publication May 10, 2002. Accepted for publication October 21, 2002.
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) and SLC antagonist-treated GVHD
mice (•) were determined by flow cytometry at several time points
during the first 16 days after parental spleen cells transfer. Values
are shown as mean ± SD (n = 4 mice/group/time
point). Similar results were observed in two additional experiments.
The statistical analysis was performed using Student’s
t test. *, p < 0.05 vs control
PBS-treated GVHD mice.
), i.v. injection of 60 µg/mouse twice, 2 and 6 h
after GVHD induction. Values are shown as mean ± SD
(n = 4 mice/group/time point). Similar results were
observed in two additional experiments. The statistical analysis was
performed using Student’s t test. *,
p < 0.05 vs in vivo control PBS-administrated GVHD
mice.