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* Rheumatology Unit, Guy’s, King’s and St. Thomas’, School of Medicine, London, United Kingdom;
Department of General and Vascular Surgery, Guy’s and St. Thomas’ Hospital Trust, St. Thomas’ Hospital, London, United Kingdom;
MediCity Research Laboratory, University of Turku, Turku, Finland;
BHF Cardiovascular Medicine Unit, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, London, United Kingdom; and
¶ Department of Biochemical Pharmacology, William Harvey Research Institute, Charterhouse Square, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and does not rely on the up-regulation
of adhesion molecules (such as ICAM-1) on the graft vascular
endothelium. This is the first description of huPLN transplantation
into SCID mice and of the functional effects of SDF-1 in regard to the
migration of human cells into huPLN in vivo. This model provides a
powerful tool to investigate the pathways involved in cell migration
into lymphoid organs and potentially to target them for therapeutic
purposes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was first
identified as an 89-aa cytokine capable of supporting the proliferation
of a stromal cell-dependent pre-B cell line (1). The amino
acid sequence of the secreted protein classified SDF-1 as a member of
the CXC chemokine (CK) family and has been recently renamed CXCL12
(2). In contrast to the majority of CK, SDF-1 interacts
specifically and monogamously with a G protein-coupled serpentine
transmembrane receptor LESTR/FUSIN subsequently termed CXCR4
(3). Binding of SDF-1 to CXCR4 on the leukocyte cell
surface results in intracellular calcium fluxes and morphological
changes associated with cell locomotion, such as the formation and
retraction of lamellipodia and integrin activation
(4). Both SDF-1 and CXCR4 expression have been found to be essential for normal myelopoiesis and lymphopoiesis both in embryo and adult life. McGrath et al. (5) have shown that the expression of CXCR4 mRNA in embryonic ectodermal structures is complementary to the expression of SDF-1 by migrating endo- and mesodermal stem cells. Defects seen in SDF-1-deficient mice suggest an important role for this CK in the recruitment and retention of leukocyte precursor lineages to myelopoietic and lymphopoietic sites (6, 7). This is also suggested by the fact that SDF-1 has been shown to be instrumental in mobilizing hemopoietic precursors from the bone marrow into the circulation and from here into peripheral tissues (8, 9). In addition, indirect evidence for an important role of SDF-1 in regulating cell migration comes from the knowledge that SDF-1 associates with heparan sulfates on the endothelial surface (10) and stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow in vitro (11). Furthermore, SDF-1 preactivation of CD34+ cells in vitro up-regulates their migration in vivo following transfer into SCID animals (12).
In humans, on the basis of a wide expression of SDF-1in psoriatic skin and rheumatoid synovium, it has been suggested that SDF-1 is important in facilitating cell migration to inflamed tissues (13, 14, 15). On the other hand, SDF-1 is also strongly expressed within splenic red pulp and lymph node medullary cords (16, 17), suggesting that SDF-1 is also involved in lymphocyte migration to secondary lymphoid organs. However, no human data are available to directly prove this point. For this reason, to investigate directly the functional role of SDF-1 in regulating human lymphocyte migration into human peripheral lymph nodes (huPLN), we have developed a new model (reported herein) in which huPLN are transplanted into SCID mice s.c.
In the work designed to validate this model, we demonstrate that 1) huPLN can be grafted onto SCID mice with a success rate greater than 90%; 2) huPLN transplants are vascularized by mouse subdermal vessels which form anastomoses with the human graft microvascular endothelium that expresses human ICAM-1; 3) mouse-human vascular anastomoses are patent and functional as shown by the capacity to deliver mAbs and human cells to the grafts via the mouse systemic circulation; 4) a proportion of graft venules retained high endothelial venule (HEV) morphology, MECA-79 and CD34 expression; and 5) markers of human lymphatic vessels were found within grafts.
In the work designed to investigate the functional role of SDF-1 in
this model, we demonstrate that 1) SDF-1 has even a greater ability
than TNF- to induce human PBL (huPBL) and U937 cell migration into
the grafts; 2) the SDF-1-induced migration was specifically inhibitable
by blocking the SDF-1 receptor CXCR4; 3) contrary to TNF-, the
SDF-1-induced migration was not dependent on endothelial up-regulation
of ICAM-1; and 4) SDF-1 and TNF- effects are independent from each
other.
To our knowledge this is the first time that huPLN have been successfully transplanted into SCID mice and that a functional effect of SDF-1 on the migration of huPBL into huPLN has been directly demonstrated. Since recirculation studies to secondary lymphoid organs in vivo in humans are impossible for obvious ethical considerations, this model offers a possible alternative approach for investigating the molecular pathways involved in regulating lymphocyte migration into lymphoid organs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Para-aortic or cervical huPLN were obtained from patients requiring vascular surgery. huPLN were of normal size and macroscopic appearance. Samples of each node were processed for routine H&E histology before their use for transplantation studies and found to have a normal histological appearance. Procedures were performed after informed consent approved by the hospital Ethics Committee (LREC 99/03/19). Samples were divided into two parts. One part was used for immunohistology and the second for transplantation. The part assigned for immunohistology was embedded in OCT compound (Miles, Torrence, CA), snap frozen in liquid nitrogen-cooled isopentane (BDH, Poole, U.K.), and stored at -70°C until analysis. The second part, assigned for transplantation, was cut into 0.5-cm3 pieces, frozen in 20% DMSO (Sigma-Aldrich, St. Louis, MO) in heat-inactivated FCS (PAA Labs, Linz, Austria), and stored in liquid nitrogen until engraftment as previously described (18). Samples of huPLN were thawed from liquid nitrogen storage immediately before surgery, washed in saline, and kept in saline-moistened sterile gauze over ice until transplanted. Beige SCID C.B-17 (NOD/LtSz-scid/scid) mice, maintained under pathogen-free conditions in biological facilities of King’s College, were anesthetized by i.p. injection of 0.2 ml Dormitor (0.1 mg/ml; GlaxoSmithKline, Uxbridge, U.K.) and 0.1 ml ketamine (0.1 mg/ml; SmithKline Beecham). A small incision was made in the dorsal skin behind the ear of each SCID mouse (4–6 wk of age) and the tissue was inserted s.c. The wound was closed with soluble suture material (Ethicon, Edinburgh, U.K.). Successful tissue transplantation was assessed before migration studies by immunohistology after 4–5 wk. This particular strain of mice was chosen to minimize this possibility that huPBL could be killed by mouse NK cells in their systemic circulation. NOD/LtSz-scid/scid mice are specifically bred not only to produce no T or B cells, but also to have no NK activity (although the animals retain nonfunctional NK cells).
Assessment of graft viability
Graft viability was assessed before immunohistochemical or morphometric analysis both macroscopically and by microscopy of H&E-stained acetone-fixed cryostat sections. Grafts judged to be necrotic or those comprising tissues other than those transplanted (e.g., murine skin and muscle) were excluded from the study.
Assessment of human vasculature within grafts
To confirm the conservation of human vasculature-associated cell adhesion molecule (CAM) and to assess the modulation of CAM expression following cytokine/CK stimulation of the grafts, we assessed the expression of human ICAM-1, VCAM-1, and E-selectin pre- and posttransplantation using species-specific mAb and standard immunohistochemical techniques (see below). The relative expression of CAM was quantified using an arbitrary scale of staining intensity from 0 to 4, where 0 indicated no staining and 4 indicated maximal staining. To determine whether the human transplant vasculature remained patent and connected with the murine vasculature infiltrating the grafts, transplanted mice were injected i.v. with either biotinylated anti-human ICAM-1 or a biotinylated isotype-matched control Ab (MOPC21). Mice were killed after 10 min and the transplants were embedded in OCT and snap frozen. Cryostat sections were then incubated with avidin-biotin alkaline phosphatase (AP) complex for 30 min followed by development using a Vector Red substrate kit. Sections were subsequently incubated with FITC-conjugated anti-human VWFVIII (Serotec, Oxford, U.K.) to identify human blood vessels and, therefore, determine the site of localization of the anti-ICAM-1 and control Abs. Sections were mounted in aqueous mountant (Immunofluor; ICN, Basingstoke, U.K.) and examined by UV fluorescence microscopy.
Quantification of human and murine vasculature
Cryostat sections of the transplants were immunostained for both
human vascular markers (including VWFVIII, MECA-79, CD34, and CCL21)
and mouse vessels using anti-murine CD31 (for details of Abs used,
see Table I
). Standard immunoenzymatic histochemistry methods
(streptavidin-alkaline phosphatase; DAKO, Glostrup, Denmark) were used
as described below. The volume fraction (Vv) of murine and human
vessels within the transplanted tissues was determined morphometrically
using a point counting technique (19). Briefly the number of
point-sampled intercepts overlying the positively stained vessels was
determined on serial cryostat sections using a light microscope
equipped with a 5 x 5 eyepiece graticule. The ratio of positive
hits to total possible hits was calculated to give the volume fraction
of vessels per equivalent volume of transplant tissue. For each
transplant, 
60 microscope fields from three cutting levels were
examined.
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Indirect staining was performed using standard techniques as previously described (20). Briefly, 10-µm acetone-fixed cryostat sections of the grafts after incubating with appropriate nonimmune serum for 20 min at room temperature to block nonspecific binding were incubated with primary Ab for 1 h. Sections were washed (50 mM TBS, pH 7.6) and incubated with the secondary Ab for 30 min. If the secondary was conjugated to biotin, sections were washed and incubated with avidin-biotin-AP complex (DAKO) for 30 min. Sections were developed using the alkaline phosphatase substrate kit Vector Red containing 1 mM levamisole to inhibit endogenous alkaline phosphatase activity. Finally, sections were washed, counterstained with Meyer’s hematoxylin, dehydrated through graded ethanols, cleared in xylene, and mounted under DePeX (BDH) for examination by light microscopy.
Immunohistochemical analysis of SDF-1 and TNF- in original lymph
nodes and grafted tissue
SDF-1 tissue distribution was analyzed immunohistochemically
using Abs specific for human SDF-1 and quantified using the same
grading score described above. The tissue distribution of TNF- was
determined immunohistochemically using a modification of the method
described by Ulfgren et al. (21). Briefly, sections were
fixed for 20 min in TBS containing 2.5% paraformaldehyde (pH 7.6,
4°C) and washed in TBS plus 0.1% saponin (Sigma-Aldrich) for 20 min.
All subsequent Ab incubations and washes were conducted in the presence
of 0.1% saponin. A standard two-layer immunoalkaline phosphatase
technique was used with a murine anti-TNF- primary and goat
anti-mouse AP-conjugated secondary Ab and vector red substrate for
visualization (for details of Abs, see Table I
). TNF-positive cells
showed characteristic intracellular distribution with a predominantly
supranuclear cytoplasmic localization as previously described
(21). Sections of rheumatoid synovium were used as a
positive control.
Cell preparation, culture, and labeling.
U937 cell culture and analysis of CXCR4 and LFA-1 expression by
flow cytometry.
The U937 human myelo-monocytic cell line was cultured in RPMI 1640
medium plus 10% FCS. Cells were subcultured and maintained at
0.5–1.0 x 106 cells/ml. For FACS analysis,
U937 cells were resuspended in PBS (0.5 x
106 cells/ml), and 100-µl aliquots were added
to a 96-well flat plate in triplicate for each treatment and incubated
on ice. CXCR4 and LFA-1 expression was assessed by addition of specific
mAbs (mAbs, see Table I) at their previously determined saturating
concentration. Nonspecific binding was minimized by the addition of
human IgG (6 mg/ml). Plates were incubated at 4°C for 1 h. Cells
were washed and incubated with F(ab')2 of goat
anti-mouse IgG-FITC before further washing and analyzed by flow
cytometry (BD Biosciences FACScan II analyzer; BD Biosciences, Mountain
View, CA).
huPBL. HuPBL were isolated from peripheral blood using Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway) density gradient centrifugation as described previously (22). The isolated huPBL were suspended in tissue culture medium (10% heat-inactivated FCS (PAA) in RPMI 1640 (Life Technologies, Grand Island, NY)) and then incubated overnight in plastic flasks to deplete adherent monocytes. The remaining nonadherent cells, mostly lymphocytes as demonstrated by FACS analysis (see below), were then washed and resuspended in serum-free PBS ready for labeling.
Cell labeling. U937 cells or huPBL were incubated with PKH26 dye (Sigma-Aldrich) at room temperature at a concentration of 100 µl of dye per 20 x 106 cells in 5 ml of diluent for 2 min before stopping the reaction with the addition of heat-inactivated FCS. The cells were then washed twice to remove unbound dye and resuspended in sterile PBS (pH 7.6) at a cell concentration of 50 x 106 cells/ml. Cell viability was determined via trypan blue exclusion and always found to be >95%. PKH26 labeling efficiency was confirmed before transplantation by examining a wet preparation of labeled cells under the UV microscope
In vivo cell migration assays
PKH26-labeled U937 cells or U937 cells blocked with Ab
anti-CXCR4 (5 x 106 cells/animal), as
described above, were injected i.v. into the tail vein of the
transplanted SCID mice in a 100-µl dose volume. SDF-1 (PeproTech,
Rocky Hill, NJ), TNF- (Genzyme, Cambridge, MA), or saline was
injected intragraft at the same time. In a separate experiment,
PKH26-labeled huPBL were injected i.v. into the tail vein of
transplanted SCID mice (5 x 106
cells/animal in a 100-µl dose volume). As above, SDF-1 or saline
was injected intragraft. The migration of U937 cells or huPBL into the
grafts was assessed histologically by UV fluorescence microscopy (see
below).
Quantification of cell migration into grafts
Samples of lymph nodes were stored at -70°C until analysis.
Serial cryosections (10 µm) were mounted on Vectabond Reagent (Vector
Laboratories, Burlingame, CA) coated slides and dried overnight at room
temperature. Sections for immunohistochemical analysis were fixed in
acetone at 4°C for 10 min, wrapped in aluminum foil, and stored at
-70°C until further use. Sections assigned for analysis of
PKH26-positive cells were washed for 10 min in PBS (pH 7.6) and mounted
using aqueous mounting media (Immunofluor; ICN). Sections were analyzed
using a fluorescence microscope (Olympus BX60; Olympus, Melville, NY).
To obtain an accurate representation of the number of PKH26-positive
cells present in the lymph node grafts, three different sections were
taken from a different cutting level (i.e., from the top, bottom, and
middle of the transplant) of each graft. The results were expressed as
the average number of cells identified in each section per high-power
field (x40 objective). On average, 100 high-power fields were
counted per transplant.
EA.hy926 culture conditions and transmigration assay
EA.hy926 cells were provided by Dr. C.-J. Edgell (Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, NC). EA.hy926 cells are a hybridoma between HUVECs and the epithelioma A549 and retain most of the features of HUVEC, including the expression of endothelial adhesion molecules and human factor VIII-related Ag (23). EA.hy926 cells were cultured in DMEM-F12 supplemented with 10% FCS and antibiotics (cultured medium) and subcultured every 3 days. The transmigration assay was performed using a protocol modified from a previous study (24). The inserts of 8-µm Biocoat 24-well plates (Stratech Scientific, Oxford, U.K.) were coated with 0.5 ml of a human fibronectin solution (50 µg/ml in PBS to give a final concentration of 5 µg/cm2) for 1 h at room temperature. After washes in PBS, EA.hy926 cells were added (5 x 104 cells in 500 µl of cultured medium) and cultured for 48 h. U937 cells, previously demonstrated to be strongly positive for the CXCR4, were cultured as described below and added at 1.5 x 106/well on top of the inserts, while 0–50 ng/ml SDF-1 was added in the bottom compartment. After 18 h at 37°C in 95% air/5% CO2, cells that had migrated through the filters were retrieved from the lower compartment and counted using a Neubauer hemacytometer following staining in Turk’s solution. In some experiments, anti-CXCR4 mAb was preincubated with U937 cells for 30 min before cell addition into the Transwell. Data are reported as the mean number of cells ± SEM migrated per well. Three experiments were performed in triplicate.
Statistical analysis
In the in vitro transmigration assay, data are reported as the mean number of cells ± SEM migrated per well. Three experiments were performed in duplicate or triplicate analyzed by ANOVA,followed by the Bonferroni test for post hoc comparisons. A p value <0.05 was taken as significant. In the in vivo cell migration assays, results are expressed as mean ± SEM unless otherwise indicated. Nonparametric statistical analyses were performed using the PC analysis package SigmaStat 2.0 (Jandel, San Rafael, CA). Initially, either the Kruskal-Wallis nonparametric ANOVA or one-way ANOVA was performed. Post hoc significance testing was conducted using Dunn’s multiple comparison tests for nonparametric data or Dunnett’s test for parametric data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish whether huPLN could be successfully transplanted into
SCID mice, 4 wk posttransplantation (optimal time determined for
synovial tissues (18)) grafts’ viability was examined
macroscopically and microscopically as described in Materials and
Methods. As seen in the representative example shown in Fig. 1, huPLN were macroscopically healthy
being fed by mice subdermal vessels with no macroscopic evidence of
inflammation observed. By macroscopic assessment, transplantation was
successful in >95% of cases. Microscopic analysis showed in some
cases evidence of tissue necrosis but in 90% of cases the
transplant tissue appeared viable with no evidence of murine cell
infiltration or signs of chronic inflammation. In subsequent
experiments, one animal from each batch was sacrificed before migration
studies to assess graft viability as above. No evidence of
graft-versus-host or host-versus-graft disease was observed in any of
the transplanted animals.
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) was compared with
pretransplant tissue on routine H&E sections and by
immunohistochemistry using species-specific Abs to B cells (CD20), T
cells (CD3), and follicular dendritic cells (FDC; CD21). In general,
the cellularity and organization of the transplants was reduced
compared with original lymph node. In four of six transplants, B cell
aggregates (CD20+) were maintained (Fig. 2B), although smaller and reduced in cell number compared
with the original tissue (Fig. 2A). In the remaining two
transplants, a diffuse B cell distribution was observed (Fig. 2C). B cell aggregates were always found in close
association with CD21+ cell clusters (Fig. 2E). In contrast, CD21+ cells were
markedly depleted in those transplants with a diffuse B cell
distribution (Fig. 2F). CD3-positive cells were scattered
throughout the transplants (Fig. 2, H and I) with
no obvious organizational pattern compared with the original lymph node
(Fig. 2G).
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, A and B, both
human and murine vessels were present and often the blood vessels of
the two species appeared to be directly connected. To determine whether
these connections were patent and functional, transplanted mice were
injected i.v. in the tail vein with either anti human-specific ICAM-1
biotinylated or an isotype-matched control Ab biotinylated (MOPC21). As
shown in Fig. 3C, immunohistochemical detection of
anti-human ICAM-1 showed discrete staining of human vessels in the
grafts that also stained positively for human VWFVIII (Fig. 3D). This confirmed that Ab localization within the grafts
occurred via anastomosis of the murine systemic circulation and the
human graft vessels. Injection with control-biotinylated mAb produced
no staining (Fig. 3E), although blood vessels could be
clearly visualized when stained with anti-human VWFVIII-FITC (Fig. 3F). The functionality of the mouse-human vascular
connection was further confirmed by the injection of PKH26-labeled
huPBL i.v. into lymph node-transplanted SCID mice. As can be seen in
Fig. 3G, PKH26-positive cells (red fluorescence) localize to
the grafts via the human vasculature (counterstained with
anti-human VWFVIII-FITC).
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It can be seen that in the original huPLN the majority of
VWFVIII-positive human vessels (Fig. 4A) also express MECA-79 (Fig. 4C). However, the expression of MECA-79 in the grafts was
significantly reduced in comparison to the original lymph nodes as
shown by volume fraction analysis (original lymph node = 10.6
± 0.58: saline-injected transplant = 0.60 ± 0.03,
p < 0.05), despite the presence of numerous
VWFVIII-positive vessels (Fig. 4B). Interestingly, although
VWFVIII-positive vessels were found both in areas of lymphocytic
aggregation and diffuse cellularity, MECA-79-positive vessels were
found only in areas of cellular aggregation (Fig. 4, B and
D). There was no significant difference in the volume
fraction (Vv) of MECA-79 staining between SDF-1/TNF- stimulated
transplants vs saline-injected controls (see later).
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, E and F). In addition, we
analyzed the expression of CCL21. CCL21 was diffusely distributed in
the T cell and subcapsular areas with an apparent association with
lymphatic vessels but only modest staining of vascular structures (Fig. 4G). The tissue distribution was similar in the transplant
tissue in comparison to original lymph nodes but with markedly reduced
staining intensity (Fig. 4H).
Finally, to investigate whether human lymphatic vessels were retained
within the grafts, we examined the expression of a cellular marker
recognized by a new mouse anti human mAb (clone 3-155, see Table I) (26). Expression of 3-155
was clearly detectable in lymphatic vessels independently from
VWFVIII-positive blood vessels both in the original huPLN (Fig. 4I) and huPLN grafts (Fig. 4J). The mAb 3-155
does not appear to cross-react with murine lymphatic vessels as we
found no staining at the periphery of the transplants or in the
surrounding subdermal mouse tissue.
SDF-1 induces U937 and PBL migration into huPLN transplanted
into SCID mice
To investigate the capacity of SDF-1 to induce human cell
migration into huPLN transplants, we injected SDF-1 intragraft and
initially examined its ability to induce graft localization of U937
cells injected into the tail vein of the animal at the same time. This
cell line was chosen because it expresses high levels of CXCR4 (Fig. 5A), the selective SDF-1
receptor, making it a useful tool to investigate the system. In
addition, we also confirmed in vitro the capacity of U937 to migrate in
response to SDF-1 using a Transwell model in which microporous filters
were coated with the EA.hy926 endothelial hybridoma cell line (see
Materials and Methods). The results, shown in Fig. 5B, demonstrate that SDF-1 caused a dose-dependant increase
in U937 migration, which was significantly inhibited by preincubation
of the cells with a CXCR4-specific mAb.
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A). These
results were compared with the level of migration induced by TNF-
(200 ng/graft, optimal dose previously determined) as a positive
control) and that of saline (negative control). It can be seen that
SDF-1 was more effective than TNF- at up-regulating U937 cell
migration, causing 3-fold increase in the number of migrating cells
compared with saline-treated animals and a 2-fold increase over the
TNF--injected group. Furthermore, SDF-1-dependent migration was
completely inhibited by the anti-CXCR4-specific mAb.
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B indicate that the
increase in migration of huPBL in response to SDF-1 vs saline baseline
migration was of similar magnitude to that observed with SDF-1-induced
U937 migration. However, huPBL showed a greater capacity than U937 to
localize to the transplants both in unstimulated and stimulated
conditions. This is indicated by the number of cells per high-power
field detected (baseline migration: huPBL, 10.1 ± 2.03 vs U937,
3.49 ± 0.40; SDF-1-induced migration: huPBL, 29.3 ± 4.12 vs
U937, 11.1 ± 1.02). Mechanisms of action of SDF-1 in inducing U937 and PBL migration into huPLN transplanted into SCID mice
To investigate potential mechanisms of action of SDF-1 in relation
to TNF-, we concentrated on three aspects: first, we explored
whether the effects of SDF-1 were mediated via the inductions of
TNF- and vice versa. Second, we examined the variation in ICAM-1
expression and the relationship of this to the degree of migration of
U937 cells and PBL into the grafts. Third, we analyzed the degree of
human and mouse vascularity in treated and untreated transplants.
SDF-1 and TNF- do not induce reciprocal expression in
huPLN grafts.
SDF-1 was diffusely distributed throughout the tissue of the
SDF-1-injected grafts with staining of the vascular endothelium,
transplant stroma, and interstitium. A similar pattern of distribution
was seen in the TNF- and saline-injected grafts, but with greatly
reduced staining intensity (saline, 1.68 ± 0.09; SDF-1, 2.26
± 0.04*; TNF, 1.84 ± 0.12; mean ± SEM of four transplants
per group examined). TNF- immunoreactivity was detected in both the
original preimplantation lymph node and in all treatment groups
posttransplantation, with typically 5–10 positive cells per square
centimeter of tissue. TNF-injected transplants showed a faint diffuse
staining reaction in addition to the discrete cellular localization
seen in the positive controls and other treatment groups. However,
there was no significant difference in cellular TNF- expression when
comparing any of the treatment groups with either control
(saline-injected) transplants, SDF-1-injected groups, or original
preimplantation tissue. This strongly indicates that human SDF-1 and
TNF- expression is regulated in a nonreciprocal fashion similarly to
published data in mice (see Discussion) (27).
In addition, this suggests that the mechanisms by which they increase
cell migration into the grafts are independent from each other.
SDF-1-induced cell migration is independent of CAM
up-regulation.
To investigate the mechanisms of action of SDF-1- vs TNF--induced
cell migration, we examined the variation in endothelial CAM expression
and the relationship of this to the degree of U937 and PBL migration
into the grafts. Immunohistochemical analysis showed a significant
increase of ICAM-1 expression in TNF--injected graft vessels (Fig. 7A), confirming our previous
results (18). However, intragraft injection of SDF-1 did
not cause a statistically significant increase in vascular ICAM-1
staining intensity compared with saline-injected controls (Fig. 7B). Despite this, it is possible that the constitutive low
level of expression of ICAM-1 within the graft vasculature may still be
responsible for the SDF-1-induced migration. A low level of E-selectin
and VCAM-1 expression was detected in the grafts. Once again, this was
not modified by SDF-1. Expression was not detectable
posttransplantation in any of the treatment groups. This result may
reflect the short kinetics of expression of E-selectin in response to
TNF or SDF-1 stimulation. VCAM-1 was expressed at low levels in the
transplants and once again this was not significantly modified by SDF-1
or TNF (data not shown).
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To determine whether the observed up-regulation in cell migration
following intragraft injections of SDF-1 or TNF- were simply due to
an increase in the vascular beds feeding the graft, cryostat sections
of the transplants were immunostained for human vasculature
(anti-VWFVIII) and mouse vasculature (anti-murine CD31). The
volume fraction of murine and human vessels within the transplants was
determined by point counting (see Materials and Methods).
There was no significant difference between the treatment groups either
in the total human endothelial surface (saline, 5.50 ± 1.82;
SDF-1, 5.97 ± 0.91; TNF, 3.78 ± 0.43; mean ± SEM of
four transplants per group examined) or mouse vascularity (saline,
10.78 ± 0.51; SDF-1, 11.12 ± 0.28; TNF, 11.01 ± 0.56;
mean ± SEM of four transplants per group examined). In addition,
scatter plots of Vv fraction vs PKH26 migration showed no discernible
pattern of association of the two variables. Transplant vascularity
showed no correlation with U937 or huPBL migration (Spearman’s rank
correlation, p > 0.05 throughout) when either
individual transplants or treatment group means were compared,
indicating that the degree of migration is largely independent of the
extent of graft vascularity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Lymph node transplantation was successful in >90% of cases, with grafts remaining viable following revascularization by mouse subdermal vessels. This was confirmed both macroscopically and microscopically. The histological appearance of transplants, when compared with original preimplantation lymph nodes, showed a number of discernible morphological changes. There was a decrease in cellularity of the grafts, accompanied by a loss of organization primarily in the T cell areas but also apparent as a reduction in the number and size of B cell aggregates observed in the transplant tissue. This was mirrored by a reduction in the number of CD21+ FDC. However, where follicular aggregates of B cells were present, they were always associated with clusters of CD21+ FDC. These CD21+ cells probably represent a resident population of FDC constitutively producing B cell-supportive factors such as B cell chemoattractant 1 (28), thus maintaining a basal level of B cell organization in cellular aggregates. The morphological changes observed in the T cell population in the grafts, on the other hand, may result from the disconnection of the lymph node transplants from the human lymphatic circulation. The reduced influx of human interdigitating dendritic cells, similarly to that reported in experimental animals following ligation of afferent lymphatic vessels (29), would be a possible explanation for the disorganization of T cells observed in our model.
Analysis of human and murine vasculature within the grafts revealed histological evidence of anastomosis formation between the two species. The anastomoses were patent and functional as shown by the localization of anti-human ICAM-1 mAb to the grafts via the mouse systemic circulation following its injection into the SCID mouse tail vein. Anti-human ICAM-1 mAb was found to bind to the luminal surface of human blood vessels, confirming that the human vasculature also maintains the capacity to express human CAM. More importantly, we demonstrated that this model is a viable delivery system of human cells to the human grafts in vivo. This is similar to our previous work in which we transplanted synovial tissue and to which we make reference regarding the optimization of experimental conditions; including number of human cells reaching the grafts, time course, and dose responses (18).
Since the ligation of afferent lymphatics in the experimental model mentioned above (29) caused disruption of the morphological and molecular features of murine lymph node HEV, we examined the expression of PNAds in the transplant vasculature. PNAds are a group of sialylated, fucosylated, sulfated glycoproteins expressed on HEV in human lymph nodes and at sites of chronic inflammation (25, 30, 31). MECA-79-positive vessels persisted following lymph node transplantation, although the volume fraction of the stained area was significantly reduced when compared with the original tissue. The distribution of MECA-79 in transplanted lymph node was mainly associated with lymphocytic aggregates but not with a diffuse cellular infiltrate underlining the importance of the cytoarchitecture and the actual presence of certain cell types and their products (e.g., lymphotoxin) in the maintenance of specialized lymphoid features (28). In addition, some of the graft vessels expressed CD34. Interestingly, despite the considerable cell loss within the grafts in comparison to the original tissue, presumably related to the lack of afferent lymph, some human lymphatic vessels appear to be preserved as assessed by the expression of the 3-155 marker. Future experiments will address the functional status of the graft lymphatic vessels (patency and potential anastomoses with mouse subdermal lymphatics) and their importance in the maintenance of huPLN transplant morphology. This model may also allow investigating the factors involved in the maintenance of HEV morphology and lymphoid aggregates in human lymphoid organs.
The other important aspect that was addressed in this article was the question of the actual functional capacity of SDF-1 of inducing lymphocyte migration to lymphoid organs in vivo in humans. The literature is rich in indirect evidence in support of this hypothesis: first, SDF-1 is expressed on the luminal surface of HEV in peripheral lymph nodes (32). Second, SDF-1 is thought to be important in mobilizing hemopoietic precursors from the bone marrow into the circulation and from there into peripheral tissues (8, 9). Third, SDF-1 associates with heparan sulfates on the vascular surface (10) and stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow in vitro (11) and in animal models (12). However, direct in vivo evidence in humans was still missing. The main reason for this is that human recirculation studies would be very difficult and unethical. The model described herein overcomes these problems and provides the first direct demonstration in vivo of the functional capacity of SDF-1 to induce human cell migration into huPLN grafts. The SDF-1-induced migration was specifically mediated by CXCR4 as demonstrated by the ability of a blocking mAb to inhibit migration of U937 cells to the grafts. Although in this article we have not formally proved this for human PBL, it is known from the published literature that the majority of circulating monocytes, B cells, and naive (CD45RA+L-selectin+) T cells are CXCR4+ and SDF-1 responsive (33). This, taken together with the recently published work of Kollet et al. (34), who demonstrated the functional capacity of SDF-1 to mediate the migration of CD34+CD38-/lowCXCR4+ human hemopoietic stem/progenitor cells to the bone marrow of SCID mice and that this effect was inhibitable by preincubation of cells with anti-CXCR4 mAbs, leads us to believe that the same would be true of our experimental model.
Finally, we demonstrated that SDF-1 was more effective than TNF-
causing approximately a 2-fold increase in migration over the
TNF--injected group. Although the experiments described herein do
not allow us to determine the proportion of injected cells which
ultimately localize to the grafts, we have made such an estimate in our
previously published work on cell migration to synovial transplants in
SCID mice using radiolabeled and fluorescently labeled cells
(18). A comparison between the numbers of cells per
high-power field in the lymph node grafts vs those found in synovial
grafts of previous experiments indicates that the proportion of
migrated cells is comparable. Therefore, considering that in both
models the number of human PBL injected i.v. was the same (5 x
106/animal), it can be deduced that the
percentage of cells localizing in the lymph node grafts is similar to
the one that localizes into synovial grafts (3–7%).
To investigate the mechanisms of action of SDF-1 in comparison to
TNF-, we first considered the level of expression of endothelial
adhesion molecules. As previously reported (19), TNF-
strongly up-regulated ICAM-1 expression; however, no up-regulation was
detected in the SDF-1- injected grafts. This suggested that the
SDF-1 effects were mediated independently of an increased
transcription/mobilization of endothelial surface ICAM-1. Therefore,
the likely explanation for our observation is that SDF-1 may be acting
in the huPLN-SCID chimera model via the classical CK mechanism, namely,
the activation of surface integrins (4, 12) of the human
cells circulating within the grafts. Activated integrins would then be
able to bind more avidly to endothelial ICAM-1 expressed at basal
level. Indirect evidence for this comes from our in vitro experiments
that confirmed that SDF-1 could induce transmigration of U937 even when
using nonstimulated endothelial cells that express ICAM-1 at basal
level (35).
To make sure that some of the effects of SDF-1 were not mediated by
the induction of TNF-, we examined their tissue distribution in all
experimental conditions. As expected, SDF-1 injection into the grafts
caused an increase in detectable SDF-1-specific immunoreactivity
compared with the saline controls but not of TNF-. Reciprocal
results were obtained when the grafts injected with TNF- were
analyzed. This suggests that the mechanisms by which SDF-1 and TNF-
increase cell migration into the grafts are independent from each
other. In addition, these data indicate that TNF- and SDF1 are
independently regulated in line with the information from gene-targeted
animals, where TNF- or lymphotoxin-
deletion leads to BLC,
SLC, ELC deficiency but has no effect on SDF-1 production
(27).
Finally, to exclude the possibility that a variable degree of graft
vascularization could have influenced the level of cell migration into
the grafts, we analyzed the total endothelial surface and the number of
human and murine blood vessels. We found that there was no significant
difference between SDF-1 and TNF- of saline-treated groups. In
addition, there was no correlation between the number of cells
infiltrating the grafts and the level of human or mouse vascularity.
Taken together, these observations indicate that the level of cell
localization to the transplant relates directly to the effects of SDF-1
or TNF-.
In summary, we have demonstrated for the first time that SDF-1-injected intragrafts can induce the migration in vivo of human cells into huPLN grafted in SCID mice. In addition, we have described in detail a new model that may be of great help in dissecting the specific functions of distinct molecules involved in regulating the migration of human cells into human lymphoid organs. Also, it may allow the investigation of factors involved in the maintenance of lymphoid architecture in humans.
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2 Address correspondence and reprint requests to Prof. Costantino Pitzalis, Rheumatology Unit, 5th Floor, Thomas Guy House, Guy’s Hospital, London SE1 9RT, U.K.
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor 1; CK, chemokine; huPLN, human peripheral lymph node; HEV, high endothelial venule; AP, alkaline phosphatase; huPBL, human PBL; CAM, cell adhesion molecule; Vv, vascular volume; PNAd, peripheral node addressin; FDC, follicular dendritic cell.
Received for publication September 24, 2001. Accepted for publication February 15, 2002.
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associates with heparan sulfates through the first -strand of the chemokine. J. Biol. Chem. 274:23916.1 on synovial T cells contributes to their accumulation within the rheumatoid synovium. J. Immunol. 165:3423. enhances lymphocyte migration into rheumatoid synovial tissue transplanted into SCID mice. Clin. Exp. Immunol. 122:133.[Medline]
/ 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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I. Hecht, L. Cahalon, R. Hershkoviz, A. Lahat, S. Franitza, and O. Lider Heterologous desensitization of T cell functions by CCR5 and CXCR4 ligands: inhibition of cellular signaling, adhesion and chemotaxis Int. Immunol., January 1, 2003; 15(1): 29 - 38. [Abstract] [Full Text] [PDF]
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