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Animal Model |
From the Department of Internal Medicine and the Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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vß3, a marker of angiogenesis. All vessels expressed
VAP-1, a marker of activated endothelial cells.
Finally, the grafts retained the ability to support immigration
by human leukocytes, as demonstrated by the functional capacity
to recruit adoptively transferred 5- (and -6)-carboxyfluorescein
diacetate succinimidyl ester-labeled T cells. T cells entering the
RA-SCID grafts became activated and produced interferon-
, as
detected by reverse transcriptase-polymerase chain reaction analysis.
These studies demonstrate that the RA-SCID model maintains many of the
phenotypic and functional features of the inflamed RA
synovium.
The mechanism of the immune component in RA has not been resolved, although large numbers of CD3+,CD4+ T cells are found in perivascular areas and CD3+,CD8+ T cells are distributed diffusely throughout the synovium.1,2 Recent evidence including the polyclonal nature of the CD4+ T-cell infiltrate and the nearly uniform expression of activation molecules by these cells suggest they play a role in perpetuating chronic inflammation.3 CD8+ cells in the synovium seem to be both proinflammatory and regulatory, whereas CD20+ B cells secreting immunoglobulins (Igs) are found in defined aggregates throughout the synovial tissue along with CD4+ T cells and interdigitating dendritic cells.2 The local generation of immune complexes and activation of complement have been proposed as mechanisms contributing to tissue damage and propagation of inflammation.3
Inflammatory cells in the RA joint consist primarily of
polymorphonuclear leukocytes, monocytes, and
macrophages.1,2
Few polymorphonuclear leukocytes are
localized in the RA synovium, although large numbers characteristically
accumulate in the synovial fluid, presumably because of limited
chemokine receptor expression. Numerous monocytes and macrophages are
found within the RA synovial tissue and fluid.1,2
Activated monocytes produce cytokines such as interleukin (IL)-1ß,
IL-6, and tumor necrosis factor- that stimulate production of
prostaglandins, matrix metalloproteinases, and other mediators from
synoviocytes and activate endothelial cells.1
Other
inflammatory cells, such as mast cells, have been found near sites of
cartilage erosion. Few natural killer cells have been found in the RA
synovium, however, it remains a possibility that common surface
molecules such as CD16 and CD56 used to detect natural killer cells are
down-modulated in inflamed synovial tissues.2
Synovial hyperplasia results in the formation of granulation tissue or
pannus that covers the cartilage and invades the bone.1
As
RA progresses, the normally thin synovial lining layer thickens,
primarily as a result of recruitment of myeloid lineage cells.
Angiogenesis, or the development of a network of new blood vessels is
one of the earliest histopathological alterations in the synovium and
seems to be required for synoviocyte hyperplasia.1,4,5
Whereas, all human endothelium express high levels of CD31 (PECAM-1),
recent studies suggest that only vessels undergoing angiogenesis
express the integrin vß3 or CD51/CD61.6,7
Endothelial
cells in inflamed tissue also express the activation marker,
VAP-1.8
These endothelial cells regulate the recruitment
of mononuclear cells. Thus, new microvascular formation correlates with
perivascular lymphoid infiltrates and aggregates as well as disease
activity.1,2
Rapid neovascularization of the synovium
seems to be ongoing and required to sustain the metabolic requirements
of the proliferating synoviocytes.
Previously, a number of experimental animal arthritis models, including collagen-induced arthritis in mice, rats, and rhesus monkeys; adjuvant-induced arthritis in rats; and antigen-induced arthritis in mice and rabbits have been developed to study RA.9-12 Although these models have contributed to understanding the pathogenic mechanisms involved in RA, none has reproduced all of the clinical and histopathological features of RA. This has limited the value of these animal models in developing a complete understanding of the pathogenic events in RA and also in testing novel therapeutic interventions. Therefore, mice with severe combined immunodeficiency (SCID) have been used as hosts to create disease models by transferring human cells to these animals.13,14 SCID mice lack the ability to reject allografts and xenografts and, therefore, retain human RA synovial grafts.15 Model systems in which human skin, thymus, and blood have been engrafted into the SCID host have been developed.16-19 A variety of models to study RA in SCID mice (RA-SCID) have been used.20-29 Many models are complex, involving engraftment of human RA synovial tissues or cell lines into SCID joints.20-22 Other models have placed synovial fibroblasts and cartilage under the renal capsule,15,23 or in sponges along with bone and cartilage.15,24-28 Although providing valuable information, many of these models do not recapitulate the multicellular and chronic nature of rheumatoid inflammation. We have therefore used a simplified RA-SCID model to examine the components and duration of disease.29,30 This model involves engrafting intact pieces of rheumatoid synovium subcutaneously in SCID mice. The current report details the phenotype and function of the major cellular components of the rheumatoid synovium in this RA-SCID model. These studies demonstrate that the RA-SCID grafts were rapidly vascularized and maintained the characteristics of the inflamed rheumatoid synovium after prolonged engraftment. Therefore it provides a unique model system to examine the influence of the RA microenvironment on the major components of the inflammatory process.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Monoclonal antibodies (mAbs) directed against human-specific
epitopes did not cross-react with murine molecules and anti-murine mAbs
did not cross-react with human epitopes. Antibodies directed against
human mononuclear cell and endothelial cell epitopes included anti-CD3
mAbs [OKT3 (American Type Culture Collection, Rockville, MD) and UCHT1
(Pharmingen, San Diego, CA)], anti-CD4 mAbs [OKT4a (Ortho
Diagnostics, Raritan, NJ) and MT310 (DAKO Corporation, Carpinteria,
CA)], anti-CD8 mAbs [OKT8 (American Type Culture Collection) and DK25
(DAKO)], anti-CD19 mAb (HD37, DAKO), anti-CD20 (B-Ly1, DAKO),
anti-CD68 (Y1/82A, Pharmingen), anti-CD45RA (2H4; the generous gift of
Dr. Chikao Morimoto, Dana-Farber Cancer Institute, Boston, MA),
anti-CD45RO (UCHL1, DAKO), anti-CD31 [JC/70A (BioGenex Laboratories,
Inc., San Ramon, CA) and MBC78.2 (Caltag Laboratories, South San
Francisco, CA)], anti-CD54 (R6.5; generous gift of Dr. Robert
Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT),
anti-CD62E (68-5H11; Pharmingen), anti-CD62P (S12; generous gift of Dr.
Rod McEver, The University of Oklahoma Health Science Center, Oklahoma
City, OK), anti-CD106 (1G11; the generous gift of Dr. Dorian Haskard,
RPMS Hammersmith Hospital, London, UK), anti-vß3 (LM609; the
generous gift of Dr. David Cheresh, The Scripps Research Institute, La
Jolla, CA), anti-collagen IV (MAB1430; Chemicon International, Inc.,
Temecula, CA), and anti-VAP-1 (2D10; generous gift of Dr. Sirpa
Jalkanen, Medicity Research Laboratories, University of Turku, Turku,
Finland). Isotype controls included MOPC (American Type Culture
Collection), X39 (Sigma Chemical Co., St. Louis, MO), and R35-95
(Pharmingen). Murine vascular endothelial cells were detected using a
rat anti-murine mAb (MECA32, Pharmingen) and anti-murine CD18 (M18.2;
generous gift of Dr. Akira Takeshima, University of Texas Southwestern)
and isotype controls. Proliferating cells were assessed with an
anti-human proliferating cell nuclear antigen (PCNA) mAb (PC10,
Pharmingen).
Tissue and Cell Preparation
Synovial tissue from RA patients who met the 1987 American College of Rheumatology criteria for classification31 was obtained from the knee or hip after joint replacement surgery. The samples were either immediately engrafted into mice, or snap-frozen in liquid nitrogen for immunohistochemical or molecular analysis. Peripheral blood mononuclear cells were obtained from normal age-matched donors.32 For some experiments, T cells were enriched by passage over either a nylon-wool column or negative-selection column (R&D Systems, Minneapolis, MN). Samples were obtained after informed consent according to the guidelines of the Institutional Review Board of University of Texas Southwestern Medical Center.
RA-SCID Mice
The protocols for the care and use of animals were approved by the
University of Texas Southwestern Medical Center Institutional Animal
Care and Research Advisory Committee. Homozygous SCID (CB.17 scid/scid)
mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and
maintained in a specific pathogen-free facility without prophylactic
antibiotics. Similar results were obtained in confirmatory experiments
performed with SCID.NOD mice
(NOD/Lts-Prkdzscid, The Jackson Laboratory)
defective in both innate and adaptive immunity.33
T-cell
deficiency was verified by staining blood for murine CD3 with mAb
(clone 145-2C11, Pharmingen) and flow cytometric analysis. Fresh
rheumatoid synovium was cut into small pieces of similar macroscopic
characteristics while in a Petri dish containing 
5 ml of RPMI 1640
on ice. Immediately afterward, the tissue was engrafted into mice.
Three to five female mice per experimental sample were grafted. Mice
were between the ages of 4 and 8 weeks old. Mice were anesthetized with
a mixture of ketamine, xylazine, and acepromazine and 0.1–0.2
cm3
pieces of RA synovium were surgically implanted
subcutaneously into the dorsum. An incision was made along the midline
and the grafts were implanted remote from the incision site. All
procedures were performed in the surgical suite of the barrier
facilities. In some experiments, mice underwent similar surgery except
that grafts were omitted and these mice served as sham-operated
controls. After 3, 6, or 12 weeks the grafts were harvested and
immediately snap-frozen. Grafts were dissected from murine tissue. They
were clearly distinguishable from surrounding murine tissue. In
addition, blood samples were collected at the time of sacrifice or at
various times after engraftment as detailed in the text. For some
experiments, enriched human peripheral blood T cells (5 to 10 x
107
in 0.1 ml of RPMI) were injected into the
tail vein of mice that had been grafted for 3 weeks. The cells were
labeled with 5 µmol/L of the fluorescent dye, 5- (and
-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before
injection into the mice as described.34
After the
indicated trafficking periods, blood samples were taken and the grafts
and organs were harvested and snap-frozen for future analysis.
Histology and Immunohistochemistry
Tissues freshly excised from SCID mice were snap-frozen in liquid nitrogen. For immunohistochemistry, 6-µm cryosections were prepared from OCT-embedded tissue and acetone fixed. Second step reagents for indirect staining were purchased from BioGenex, with the exception of immunohistochemistry using rat anti-mouse Ig mAbs that were developed using a polyclonal biotinylated rabbit anti-rat antibody (DAKO). Tissue sections were incubated for 60 minutes with serum to block nonspecific binding and afterward for 90 minutes with specific antibody or isotype-matched control mAb (20 µg/ml), washed, and treated for 30 minutes with hydrogen peroxide blocking solution. The samples were washed, incubated for 15 minutes with avidin blocking solution, washed, and incubated an additional 15 minutes with biotin blocking solution. Subsequently, the samples were washed and then incubated for 20 minutes with biotinylated secondary antibody. The sections were washed and incubated for 20 minutes with streptavidin-conjugated horseradish peroxidase. After washing, the sections were developed with 3-amino-9-ethyl carbazole. The tissues were washed and counterstained with hematoxylin and eosin (H&E) (Sigma Chemical Co.). Photomicrographs were taken using an Olympus BHTU light microscope equipped with an Olympus PM-10AD 35-mm photomicrographic system. Unless otherwise indicated, similar staining patterns were observed for multiple sections from at least three different RA synovial tissues analyzed before and after engraftment.
Detection of CFSE-Labeled Migrating T Cells
For histological analysis of CFSE-labeled migrating T cells, cryosections were prepared from OCT-embedded tissue. Serial sections from each tissue were either stained with H&E or immediately assessed for green fluorescence and histology using a Zeiss Axiovert 100M light microscope (Carl Zeiss, Oberkochen, Germany) equipped with an AttoArc lamp, appropriate filters, and digital Axiocam imaging system. In some experiments, CFSE-labeled T cells in murine peripheral blood or control T cells cultured in the presence or absence of phytohemogglutinin (PHA) (Wellcome Diagnostics, Greenville, NC) were stained with quantum red-labeled anti-human CD3 (Sigma Chemical Co.) and analyzed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson, Mountain View, CA).
Human Immunoglobulin and IL-6 Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay for IL-6 (R&D Systems) and human Ig (The Binding Site, Birmingham, UK) were performed on serum or plasma samples according to the manufacturer’s instructions. Samples from sham-operated mice served as controls. Human IL-6 (pg/ml) or Igs (ng/ml) could not be detected in samples from sham-operated mice as minimal cross-reactivity was observed in these assays.
RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction
RA synovial samples were homogenized in guanidinium isothiocyanate
and RNA was extracted by CsCl gradient as previously
described.35
The RNA was recovered from the gradient,
dissolved in 100 µl of diethyl pyrocarbonate-treated water and the
OD260 was determined by UV spectrophotometry. For
first-strand DNA synthesis, 2 µg of RNA was converted to cDNA by
incubation with AMV reverse transcriptase (Promega). The polymerase
chain reaction reactions were performed with the indicated specific
primers under nonsaturating conditions as previously
published.35
All samples were positive for G3PDH mRNA that
was assayed to control for the integrity of the cDNA and samples were
analyzed with primers for interferon (IFN)- (Clontech, Palo Alto,
CA) and TCR-Cß.35,36
The amplification reaction was
performed under standard conditions and the samples were run on a 1.2%
agarose gel. Molecular weight markers were used to determine the
product size for IFN-, 427 bp, and for TCR-Cß, 400 bp.
Mitogen-stimulated peripheral blood T cell cDNA was used as a positive
control. Water controls were negative for the amplified products (data
not shown).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Histological changes in the grafts were studied using
immunohistochemistry on serial sections of rheumatoid synovial tissue
obtained before and after engraftment. H&E staining revealed that
freshly isolated rheumatoid synovium contained numerous infiltrating
leukocytes among the lacy connective tissue composed of loosely
organized fibroblasts (Figure 1)
. The
majority of CD3+ T cells in the freshly isolated
synovial tissue were CD4+,
CD45RO+ memory T cells primarily localized in
perivascular regions (Figure 1)
. Fewer CD8+ cells
were distributed throughout the synovial tissue (data not shown).
Similar numbers of leukocytes consisting mainly of
CD3+ T cells persisted in the RA-SCID grafts
after 3 or 12 weeks (Figure 1)
. Staining of freshly isolated tissue for
CD68 revealed that myeloid lineage cells were found predominantly at
the synovial lining and scattered throughout the graft (Figure 2)
. After engraftment, the
CD68+ cells persisted. However, the graft
underwent reorganization such that the lining layer was no longer
clearly demarcated, but rather CD68+ cells were
distributed throughout the graft. Notably, the fibroblasts within the
graft became denser and the synovium no longer retained the loose
organization of the fresh tissue. Less frequent aggregates of
CD20+ B cells were observed in the fresh tissue
and were also maintained in the grafts (Figure 2)
.
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The reorganization of the graft described above could be in part
explained by the host reaction to the implanted tissue. When viewed
macroscopically, it is apparent that the host forms a highly
vascularized fibrous capsule around the graft (Figure 3)
. In some experiments, Luconyl blue, an
intravascular dye, was administered to mice via the tail vein
immediately before sacrifice. In these mice, the continuity of the
murine and human vasculature was readily apparent as demonstrated by
the presence of the dye in the human blood vessels within the RA
synovial graft (data not shown). Immunohistochemical analysis with an
antibody specific for murine blood vessels, MECA-32, indicated that
small numbers of murine blood vessels invaded the graft. However,
murine vessels were primarily confined to the periphery in the region
of murine-human anastomosis (Figure 4)
.
As expected, no staining with MECA-32 was observed in fresh rheumatoid
synovial tissue. Conversely, staining with an antibody for activated
human endothelial cells (VAP-1) demonstrated specific staining of
vessels within the graft, whereas no staining was observed in the outer
murine fibrous capsule (Figure 5)
.
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. Consistent with vessels at sites of
inflammation, both large and small vessels expressed the endothelial
cell activation molecule, VAP-1. Of note, vß3, a receptor
transiently up-regulated on endothelial cells during angiogenesis was
localized to small vessels throughout the synovium. A similar pattern
of staining for expression of blood vessels was observed on grafts
after 3, 6, or 12 weeks (Figure 7)
. Both
large and small vessels were detected with CD31 at each time point.
Importantly, staining with the endothelial cell activation marker,
VAP-1, revealed that the endothelium remained activated for up to 12
weeks after engraftment, expressing VAP-1 in a similar manner to
freshly derived tissue. Notably, intense staining with vß3 was
observed on small vessels throughout the grafts, even after 12 weeks of
engraftment, indicating that angiogenesis remained an ongoing active
process in the grafts. Staining for the proliferation marker, PCNA,
confirmed that new vessel formation in the inflamed tissue continued
after engraftment into the SCID mice (Figure 8)
. Fibroblasts in the grafts also
expressed PCNA. These findings suggest that the human vasculature
within the graft retained the proinflammatory features of the freshly
isolated synovium and indicated that angiogenesis of human vessels was
an active ongoing process in the grafts.
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To demonstrate that the grafts in the RA-SCID mice maintained the
inflammatory elements of freshly isolated tissue, Ig and IL-6 were
assayed in the SCID mouse serum after 3 and 12 weeks of engraftment. As
expected, minimal cross-reactivity was detected for human Ig and human
IL-6 in serum from control nonengrafted mice (Figure 9)
. In contrast, human Ig and IL-6 were
detected in the sera samples from mice that had been engrafted with RA
synovium for 3 and 12 weeks. In most cases, IL-6 levels declined
throughout the course of the experiment, unless the mouse received T
cells or other proinflammatory signals (Figure 9)
. In addition, the
presence of various subclasses of human IgG was examined in RA-SCID
mouse blood. As can be seen in Table 1
,
all subclasses of IgG were detected in the blood of RA-SCID mice. IgM
was detected, but at a lower frequency than IgG. By contrast, no human
IgG or IgM could be detected in sham-operated control samples. These
data suggest that ongoing B cell and monocyte activity persisted in the
RA-SCID grafts. Importantly, these serological assessments provide a
means to monitor ongoing inflammation and immunological activity in
this model.
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Murine leukocytes did not readily infiltrate the graft as was
demonstrated by the localization of murine CD18 to the periphery of the
graft. As can be seen in Figure 10
,
staining with anti-murine CD18 revealed no murine leukocytes in the
graft. In contrast, murine leukocytes expressing CD18 were detected in
the connective tissue surrounding the graft.
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CFSE-labeled T cells were found to reside specifically in the grafts at
45 hours after injection (Figure 11)
.
By contrast, few CFSE-labeled cells were detected in the liver, heart,
spleen, or lung of mice that carried synovial tissue grafts. In
addition, spleens of control mice seemed to have similar numbers of
mononuclear cells as spleens from injected animals. Thus, these
experiments demonstrated that human peripheral blood T cells
specifically migrated into and accumulated in the grafts.
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shows the results of grafts
harvested from five different mice and the T-cell-specific mRNA
contents of the grafts as assessed by reverse transcriptase-polymerase
chain reaction. All lanes were positive for G3PDH (data not shown). The
results suggest that endogenous T cells persisted in the grafts (Figure 12
, lanes 1 to 3; lane 1 was weakly positive for TCR-Cß), however,
IFN- could not be detected in RNA preparations from the grafts until
after adoptive transfer of additional T cells (Figure 12
, lanes 4 and
5). Therefore, these studies indicate that RA synovial grafts promote
minimal ongoing production of IFN- by the resident T cells.
These studies also suggest, however, that either
IFN--producing Th-1 cells selectively home to the graft or that the
microenvironment provided by the graft induces Th-1 cell activation of
newly migrated T cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Some differences between previous RA-SCID models and the current model were noted. For example, in previous models in which cell suspensions or small pieces of synovium were implanted, lymphodepletion was routinely observed.15,21,38,39 In some of these models the synovial tissue was implanted under the kidney capsule or in an ear pouch. It is possible that the location of the graft played a role in the lymphodepletion. Moreover, depletion of leukocytes within the grafts was observed in our preliminary studies when we used smaller grafts or grafts consisting of fine fronds of tissue. Under these conditions it seemed that the cytokines and/or chemokines within the grafts were not sufficient to retain the leukocytes and they migrated out of the grafts. In the current experiments, the grafts were at least two to three times larger than the grafts in the preliminary experiments. In these grafts, similar numbers of leukocytes and specifically CD3+, CD4+ memory T cells persisted after 12 weeks. Thus, these data suggest that a certain mass of tissue containing the appropriate elements was required to sustain the immunoinflammatory nature of the graft. The production of human IL-6 and Ig confirmed that the B cells and macrophages retained in the grafts continued to be activated.
An important characteristic of RA is the presence of autoantibodies in the circulation. Although we have not tested for rheumatoid factor in the serum of these mice, the observation that relatively high levels of human Ig were produced in the synovium throughout the length of the experiments is significant. Despite the disruption of the B-cell follicles by proliferating fibroblasts, plasma cells continued to secrete antibodies for at least 3 months. In humans, polarized T-cell populations have been reported to bias Ig responses. Thus, Th-1 cells induce IgG1 and IgG3 whereas Th-2 cells support IgG2 and IgG4 responses. No obvious bias was observed in antibody production even though we have previously reported that T cells in the RA synovium are Th-1 polarized.32 This is likely the result of the combination of Th-1 cells and IL-13 being present in the rheumatoid microenvironment. IL-13 has similar effects to IL-4 on B cells, whereas the IL-13 receptor is absent from T cells. Thus, the RA-SCID model provides a unique opportunity for gaining insight into the local immune response that is likely the source of pathogenic autoantibodies. Current studies are focused on investigating this aspect of the model in greater detail.
Our studies suggest that ongoing angiogenesis and activation of
human endothelial cells was promoted in the grafts. We observed that
when Lyconyl blue was administered intravenously immediately before
sacrifice, the anastomosis between murine and human vasculature was
readily apparent. Others have noted similar results by dual staining
for human and murine vessels indicating ongoing
angiogenesis.39
The establishment of an anastomosis
between murine and human vessels suggests that complex recognition and
organization of structural elements might have allowed continuity of
vessels. However, it was evident that murine vessels were primarily
excluded from the human grafts, whereas human vessels did not
infiltrate the surrounding murine tissue. In the grafts, angiogenesis
was indicated by the presence of human vß3 on small vessels
identified by staining for human CD31 and VAP-1. It should be noted
that vascular endothelial growth factor was detected in the serum of
grafted mice, suggesting that angiogenic factors were produced for
weeks after engraftment (unpublished observation). Human collagen IV
was detected, using species-specific antibodies, along the basement
membranes of large and small vessels in fresh synovium and continued to
be expressed after 12 weeks of engraftment. The pattern of collagen
expression was similar in fresh RA synovium and in RA grafts
(unpublished observation). The expression of additional molecules
up-regulated on blood vessels in inflamed synovium was studied in fresh
rheumatoid synovium and in grafts. VCAM-1 (CD106) and
E-selectin (CD62E) were expressed on the vessels in RA synovium and in
RA grafts. Increased expression of ICAM-1 (CD54) was also observed on
endothelial cells in fresh RA synovium and in RA grafts. ICAM-1 was
also expressed by the synovial lining cells and mononuclear cells
scattered throughout the synovium. Although the lining layer was
disrupted in RA grafts, ICAM-1 expression was maintained on the myeloid
cells in the synovial grafts (unpublished observation). Thus, the
grafts maintained a high level of vascularity and many features of the
inflamed RA synovium were retained in the RA grafts.
It should be noted that in a similar model cellular adhesion molecules
on microvascular endothelial cells in RA-SCID grafts were
down-regulated after 4 weeks after transplantation.39
These cellular adhesion molecules, indicative of an inflammatory state,
could be up-regulated once again by intragraft injection of tumor
necrosis factor-. Several important differences were observed
between this report and the current studies in tissue preparation and
engraftment. In the previous studies, the size of the tissue used was
considerably smaller and synovial tissue was frozen and thawed before
implantation. We have observed that most mature myeloid cells are
highly sensitive to freeze and thaw techniques and might not have
survived initial engraftment. As previously discussed, we have found
that smaller grafts are unable to retain leukocytes, presumably as a
result of an inability to maintain the appropriate cytokine, chemokine,
and adhesion molecules in the more limited microenvironment.
Lymphodepletion has been reported in several similar
models.15,38,39
Likewise, Wahid and
colleagues39
concluded that in the absence of T cells
producing the required cytokines, after 4 weeks, the grafts returned to
a "resting state" and the vasculature expressed decreased levels of
ICAM-1 and VCAM-1 as compared to fresh tissue. Restimulation of the
vasculature by intragraft injection of cytokines38,39
or
intragraft injection of activated lymphocytes38
up-regulated adhesion molecules on the human vessels within the grafts.
Thus, it is likely that the prolonged expression of cellular adhesion
molecules observed in the current study directly reflects ongoing
cytokine and/or chemokine production by the graft.
The current experiments suggest that human T cells specifically migrated into the synovial grafts via the human blood vessels. Thus, human blood vessels in the grafts maintained a remarkably similar phenotype to fresh RA synovial tissue. As opposed to previous studies in which human T cells were localized to the murine fibrous capsule surrounding kidney grafts,15 we and others found that human T cells were found predominantly surrounding the vessels and distributed throughout the grafts.39 Specificity was demonstrated by the comparative absence of murine leukocytes in the grafts. Thus, the route of T-cell injection, graft size, or the location of the graft might have contributed to sufficient vascularization that allowed trafficking of human leukocytes via transendothelial migration into the grafts.
Several studies have suggested that the RA synovial microenvironment
plays a role in biasing CD4+ T cells toward a
Th-1 phenotype. Interestingly, we found that similar to previous
reports examining freshly isolated synovial T cells, resident T cells
within the RA grafts were unable to sustain detectable levels of
IFN- mRNA.40
We have previously shown, however, that
freshly isolated T cells from RA synovial tissue or fluid display a
Th-1 cytokine profile after a brief in vitro
activation.32
Moreover, a significant increase in IFN-
mRNA in circulating RA peripheral blood mononuclear cells was observed
when entry of circulating T cells was blocked with a mAb to the
adhesion molecule, ICAM-1, suggesting a redistribution of activated
Th-1 cells from the inflammatory site into the peripheral
circulation.35
These studies suggest that chronic Th-1
activation might contribute to the inflammatory state. In the current
studies, freshly isolated peripheral blood T cells rapidly
converted to IFN--producing Th-1 effector cells on entry into the
graft. In preliminary experiments, similar results were obtained with
autologous synovial tissue T cells that were maintained in culture
until injection. Although, the role of specific chemokines in
attraction and retention of Th-1 cells in the RA synovium
is controversial, the data suggest that the RA synovium either selects
by the production of specific chemokines or directs by the production
of specific cytokines the activation of Th-1-polarized T
cells.32,41,42
The current model system provides a unique
approach to dissecting the role of the synovium in biasing T-cell
responses.
It should be mentioned that throughout the course of these experiments, no overt signs of graft versus host disease were observed in the grafted animals. This was most probably because the lymphocytes were retained in the grafts. Moreover, no acute graft versus host disease was seen when T cells were injected for trafficking studies. Previous studies have suggested that without sufficient autologous antigen-presenting cells, human xenoreactive T cells become tolerant in these animals.43 To determine human T-cell reactivity to SCID mononuclear cells, we examined the response of human peripheral blood mononuclear cells to SCID spleen cells in vitro and observed minimal responses at day 3 and 6 of culture as assessed by measuring DNA synthesis (unpublished observation). The same cells responded robustly to the T-cell mitogen, PHA. Therefore, it seems that graft versus host disease by resident T cells in the graft is unlikely, because these cells are retained within the graft, respond poorly to murine stimulator cells, and appear to express little, if any, Th-1 cytokines. T cells injected for trafficking studies that do not migrate into the graft seem to undergo rapid clearance from the animal. Moreover, our preliminary data suggest that either the precursor frequency of xenoreactive T cells is fairly low or that other as yet undetermined suppressive effects of SCID spleen cells contribute to the poor stimulatory capacity of these cells.
In summary, RA-SCID grafts retain human leukocytes and produce Igs and
cytokines similar to the fresh synovial tissue. Moreover, these grafts
sustain angiogenesis and the vascular endothelium maintains
an activated phenotype. The grafts support the recruitment and
activation of T lymphocytes. Such activated T cells produce the
proinflammatory cytokine, IFN-. This model system will be used to
further understanding of the regulation of the major components of the
rheumatoid synovium and to investigate the efficacy of new therapeutic
modalities for RA.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant AR45293.
Accepted for publication October 3, 2001.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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vß3 antagonist. J Clin Invest 1999, 103:47-54[Medline]
treatment in human/murine SCID arthritis. Ann Rheum Dis 1999, 58:428-434 and mediated through ICAM-1. Clin Exp Immunol 1996, 106:20-25[Medline]
, interleukin-1ß, and activated peripheral blood mononuclear cells on the expression of adhesion molecules and recruitment of leukocytes in rheumatoid synovial xenografts in SCID mice. J Rheumatol 1999, 26:1877-1889[Medline]
) enhances lymphocyte migration into rheumatoid synovial tissue transplanted into severe combined immunodeficient (SCID) mice. Clin Exp Immunol 2000, 122:133-142[Medline]
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 R. Korn, S. Rohrig, S. Schulze-Kremer, and U. Brinkmann Common denominator procedure: a novel approach to gene-expression data mining for identification of phenotype-specific genes Bioinformatics, June 1, 2005; 21(11): 2766 - 2772. [Abstract] [Full Text] [PDF]
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 P. Maffia, J. M. Brewer, J. A. Gracie, A. Ianaro, B. P. Leung, P. J. Mitchell, K. M. Smith, I. B. McInnes, and P. Garside Inducing Experimental Arthritis and Breaking Self-Tolerance to Joint-Specific Antigens with Trackable, Ovalbumin-Specific T Cells J. Immunol., July 1, 2004; 173(1): 151 - 156. [Abstract] [Full Text] [PDF]
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B. P. Leung, M. Conacher, D. Hunter, I. B. McInnes, F. Y. Liew, and J. M. Brewer A Novel Dendritic Cell-Induced Model of Erosive Inflammatory Arthritis: Distinct Roles for Dendritic Cells in T Cell Activation and Induction of Local Inflammation J. Immunol., December 15, 2002; 169(12): 7071 - 7077. [Abstract] [Full Text] [PDF]
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