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Department of Dermatology, Case Western Reserve University/University Hospitals of Cleveland, Cleveland, OH 44106
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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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In scleroderma, overproduction of normal collagen at both mRNA and
protein levels has been shown by many investigators (6, 7). TGF-ß protein and messenger RNA have been localized to
sites of mononuclear cell infiltration and fibroblast activation by
immunostaining and in situ hybridization studies of lung and skin
tissue from scleroderma patients (8, 9). We hypothesize
that monocyte activation by host-reactive T cells is an initiating
event in scleroderma and Scl GVHD. These monocytes infiltrate skin and
produce TGF-ß1, which then causes the collagen up-regulation leading
to skin fibrosis. If this hypothesis is valid, it follows that TGF-ß1
inhibition may prevent fibrosis in early disease (Fig. 1
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven- to eight-week-old female B10.D2 (H-2d) and BALB/c (H-2d, Jackson Laboratory, Bar Harbor, ME) mice were utilized as donors and recipients, respectively, for BMT to produce Scl GVHD (10, 11, 12). Briefly, recipient mice were lethally irradiated with 700 cGy from a Gammacel 137Cs source. Approximately 6 h later they were injected i.v. by tail vein with donor spleen (2 x 106/mouse) and bone marrow cells (1 x 106/mouse) suspended in RPMI 1640 (BioWhittaker, Frederick, MD) with 10 U/ml heparin (Fisher Scientific, Pittsburgh, PA) (12). A control group of BALB/c recipient mice received BALB/c spleen and bone marrow cells (syngeneic BMT, referred to as control animals). Transplanted animals were maintained in sterile Micro-Isolator cages (Lab Products, Seaford, DE) and supplied with autoclaved food and acidified water. The dose of donor cells used in these experiments was determined from the literature (10, 11, 12), and from pilot experiments (data not shown) in which an increasing number of spleen cells produced no additional significant skin thickening.
Inhibition of fibrosis with Abs to TGF-ß
A total of 150 µg of anti-pan TGF-ß Abs (rabbit polyclonal IgG, Sigma, St. Louis, MO) or 150 µg of control rabbit IgG (Sigma) were administered by tail vein injection on day 1 and again on day 6 post-BMT. The dose was selected as a standard one used for other mouse models (13, 14, 15). Mice were sacrificed at day 21 as described below.
Collection of tissue
Three animals per group (experimental or control BMT) per time point were sacrificed via cervical dislocation at days 7, 14, 21, 38, 49, and 75 post-BMT (36–50 animals per experiment). Back skin was depilated and harvested for RNA extraction (snap-frozen in liquid nitrogen), flow cytometry, immunostaining (frozen on dry ice), and routine histologic staining (fixed in 10% buffered formalin, Surgipath Medical Industries, Richmond, IL). Tongue and lung were also harvested, fixed in formalin, and embedded in paraffin for routine staining.
Histological and morphometric analysis
Formalin fixed, paraffin-embedded sections of tissue were stained by hematoxylin and eosin (Surgipath Medical Industries). Frozen skin was embedded in OCT embedding medium (Miles, Elkhart, IN) and sectioned by cryostat (Leica CM1800, Nussloch, Germany) for immunostaining (described below). For morphometric analysis, histological sections of lung or back skin were scanned with a CCD camera (Optronics, Goleta, CA) using an Axiophot photomicroscope system (C. Zeiss, Oberkochen, Germany), stored as TIFF files, and subjected to image analysis (Optimas 6.1, Bothell, WA). Areas were calculated in arbitrary square units by outlining the dermis on a 10x view for each microscopic image, in which length was fixed and thickness varied, giving an average thickness for a broad area of skin. A minimum of eight measurements were taken from two or more skin sections from each animal. For lung tissue, a minimum of eight measurements from one lobe of each lung were taken and the percentage of alveolar space was calculated.
Antibodies
Immunostaining. Anti-CD11b (anti-Mac-1, mAb M1/70, rat IgG2b, PharMingen, San Diego, CA) was used to identify monocyte/macrophages. Goat anti-rat IgG-biotin (Vector Laboratories, Burlingame, CA) followed by Streptavidin alkaline phosphatase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and diaminobenzidine (Kirkegaard & Perry) were used for detection. Isotype control Abs were rat anti-IgG2b (R35-38, PharMingen).
Flow cytometry.
PE-labeled mAb M1/70 (PharMingen) was used to detect mononuclear
CD11b-positive cells in skin. FITC-labeled mAb to CD3
(145-2C11,
Armenian Hamster IgG, PharMingen) was used to detect T cells in
skin.
Immunostaining
Staining on frozen sections of skin was performed at least three times on each specimen by standard methods (16).
Isolation of RNA from skin
Total RNA was isolated from snap-frozen skin by guanidinium HCl method (17) and stored at -80°C until use in RT-PCR analysis and RNase protection assays.
RT-PCR for TGF-ß1
Specific oligonucleotide primers for TGF-ß1 or G3PDH (both from Clontech, Palo Alto, CA) were employed in RT-PCR reactions with total RNA from skin as previously described for analysis of other cutaneous cytokine mRNAs using Gene Amp 9600 PCR System (Perkin-Elmer, Norwalk, CT) (18). The cycle number of 30 was chosen so that the TGF-ß1 and GP3DH signals were in the linear range on ethidium bromide-stained gels, which were photographed and acquired via GelDoc (Bio-Rad, Hercules, CA). The bands were then analyzed by image analysis using Optimas 6.1 software and the results expressed as the relative density for TGF-ß1 following normalization for the RNA loading amount based on the GP3DH band.
RNase protection assays to detect pro
1(I) collagen mRNA
We prepared riboprobes from cDNAs internally labeled with
digoxigenin UTP according to the manufacturer’s instructions (Genius
II kit, Boehringer Mannheim, Indianapolis, IN). RNase protection
assays with a riboprobe for pro1(I) collagen (cDNA, a kind gift from
E. Vuorio, University of Turku, Turku, Finland), (19) were
performed according to the manufacturer’s instructions (RPAII kit,
Ambion, Austin, TX) to assay and quantify collagen mRNA expression in
mouse skin. Briefly, the gel-purified riboprobes were hybridized to
mRNA prepared from skin and digested with RNase to remove nonhybridized
sequences. The hybridization products were separated on a 5%
nondenaturing polyacrylamide gel, electrophoretically transferred to
HybondN+ nylon membrane (Amersham, Arlington Heights, IL),
and detected with peroxidase-conjugated Abs to digoxigenin (Boehringer
Mannheim) by chemiluminescence (Supersignal, Pierce, Rockford, IL).
Images were obtained by exposure to x-ray film and the results analyzed
by image analysis using Optimas 6.1 software. Collagen mRNA expression
was normalized to a ß actin or 28S rRNA control (Ambion).
Preparation of dermal cell (DC) suspensions for flow cytometry
Small pieces of depilated skin were digested in RPMI containing 10 mM HEPES (Irvine Scientific, Santa Anna, CA), 0.01% DNase (Sigma), 0.27% collagenase (Sigma), and 1000 U of hyaluronidase (Sigma) at 37°C for 2 h (20). The digested dermis was filtered through nylon mesh to generate a single cell suspension of DCs containing resident cells (fibroblasts, endothelial cells, and perivascular cells) and infiltrating cells (lymphocytes and monocytes). The cells were stained with PE-labeled mAb for CD11b (PharMingen). After fixation with 1% paraformaldehyde in PBS, flow cytometry was performed with an Epics Elite Cytometer (Coulter, Hialeah, CA) and the data were analyzed with Coulter Elite software. Analysis of CD11b+ cells was performed with and without gating to exclude polymorphonuclear cells based on light scatter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lethally irradiated BALB/c mice transplanted via tail vein injection with bone marrow and spleen cells from B10.D2 mice develop skin thickening in the setting of mild histological and clinical changes of cutaneous GVHD (Scl GVHD). Animals that do not engraft die, and control BALB/c animals receiving syngeneic transplants do not develop GVHD or skin thickening. All engrafted animals show colony formation in the spleen, while the nonengrafted animals that died lacked spleen colony formation at autopsy. GVHD is clearly present in experimental animals, seen histologically as satellitosis and apoptotic keratinocytes in tongue epithelium and skin (data not shown). However, the numbers of dyskeratotic keratinocytes in epithelium, indicating severity of GVHD, are small (21). The form of GVHD is fibrotic, rather than cytotoxic. The experimental animals show no alopecia or diarrhea, but fail to gain weight, as expected, from 8 to 18 wk following BMT.
Sclerodermatous cutaneous disease occurs in experimental animals by day 21
Sclerodermatous thickening of skin is detectable by day 21
post-BMT by image analysis of routine histopathological sections (Fig. 2A), with an increase in total
dermal area in animals with Scl GVHD of 
40% over the syngeneic BMT
control animals (Fig. 2B). We have noted skin thickening as
early as day 14, however 21 days is usually required to observe
significant skin thickening in all transplanted animals. The
variability in the earliest detectable skin thickening is most likely
due to the success of the BMT and the quality of the spleen cell and
bone marrow preparations. The skin thickening remains constant as late
as 76 days post-BMT.
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Increased TGF-ß1 mRNA was observed by semiquantitative RT/PCR
analysis of total RNA prepared from whole mouse skin (Fig. 3). Approximately 3-fold more TGF-ß1
mRNA is seen in the skin of experimental compared with control animals
as early as 6 days post-BMT. On day 21 post-BMT, TGF-ß1 is at least
2-fold higher in animals with Scl GVHD than in syngeneic BMT control
animals. Each lane represents data from one animal and is
representative of four experiments with two or three animals per group
for each experiment. Therefore, increased TGF-ß1 in skin precedes
skin thickening, as predicted by the hypothesis.
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We used an RNase protection assay to quantify collagen mRNA
production in the skin of mice with Scl GVHD. Up to 15-fold increased
collagen mRNA synthesis is seen in the skin of experimental animals
with Scl GVHD (E) compared with control (C) animals on day 38 post-BMT
(Fig. 4). The increased message is
detectable on day 21 and returns to near control levels by day 75.
Therefore, increased collagen mRNA
synthesis correlates with increased dermal fibrosis. A
digoxigenin-labeled ß-actin riboprobe added to each hybridization
solution served as an internal standard for the amount of RNA in the
hybridization reaction and the amount loaded on the gel. All collagen
densities were normalized to the ß-actin signal. For the
representative day 38 ribonuclease protection assay (RPA) gel lanes
shown, the ß-actin signals could not be equalized due to the extreme
up-regulation of the collagen mRNA signal. The calculation of 15-fold
up-regulation takes into account the differing ß-actin signals.
Similar results were obtained using a 28S ribosomal RNA probe.
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). CD11b+ mononuclear cells in cutaneous infiltrates in Scl GVHD
We performed immunostaining of frozen sections of skin, and showed
prominent infiltration of CD11b+ mononuclear cells
(brown-staining) by day 21 in the skin of animals with Scl GVHD
compared with syngeneic BMT controls (Fig. 5A). There are a few
infiltrating CD11b+ cells by day 7, they are markedly
increased by day 14, and prominent by day 21. Isotype control Ab
immunostaining is negative. The examples shown are representative of
three experiments. The infiltrates are especially dense in deep dermis,
where fibrosis typically occurs first in human scleroderma.
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B). The results correlate with immunostaining, showing
increased CD11b+ cells infiltrating the skin of
experimental animals with Scl GVHD (E) but not control
animals (C) on day 21. The scatter plots shown are
representative of three experiments. The CD11b+ cells
present were mononuclear cells, not polymorphonuclear neutrophils
(PMN), which also stain for CD11b. Histological analysis has also
confirmed that there are very few PMN infiltrating the skin of Scl
GVHD mice. The remainder of cells in the dermal suspensions used for flow cytometry studies are T cells, resident dendritic cells, fibroblasts, endothelial cells, follicular and sebaceous epithelial cells. To evaluate the T cell component in Scl GVHD, we stained the DC suspensions with labeled Abs to CD3e. CD3e+ mononuclear cells make up approximately 5–15% of the total cell population in control animals on day 21 and are approximately 1.5- to 2-fold higher in animals with Scl GVHD (data not shown). CD3e, similar to CD11b+, can also be expressed on NK cells. Therefore, the roles of T and NK cells infiltrating the skin of Scl GVHD animals will require further investigation with specific mAbs.
Inhibition of skin fibrosis with anti-TGF-ß Abs
Because TGF-ß1, a fibrogenic cytokine (23), appears
to play a central role in the development of scleroderma and Scl GVHD,
we next asked if Abs to TGF-ß could prevent skin fibrosis.
Anti-pan-TGF-ß Ab (polyclonal rabbit IgG) was administered to animals
at days 1 and 6 post-BMT followed by sacrifice on day 21. Day 21
post-BMT was chosen for sacrifice because it is the time point by which
all parameters of Scl GVHD are demonstrable, including skin thickening
and up-regulated TGF-ß1 and collagen mRNA. This dose is also within
the range of normal for Ab inhibition of TGF-ß in other mouse models
(13, 14, 15). The anti-TGF-ß1 Ab treatment did not
prevent successful BMT, as evidenced by colonization of the spleen in
both control and experimental animals, however, the blocking Ab
prevented the skin thickening seen in murine Scl GVHD (Fig. 6, A–D). Untreated syngeneic BMT
control animal skin is shown in Fig. 6A. Skin thickening
accompanied by prominent mononuclear cell infiltrates in untreated
animals with Scl GVHD is shown in Fig. 6B. Anti-TGF-ß Ab
treatment prevented skin thickening in these experimental animals (Fig. 6C and Fig. 7). Anti-TGF-ß
treatment of syngeneic BMT control animals had no effect on skin
thickness (Fig. 6D). Therefore, the administration of an
antagonist to the fibrogenic cytokine TGF-ß can prevent the cutaneous
fibrosing process in early Scl GVHD, presumably by blocking
TGF-ß.
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Because pulmonary fibrosis is a major cause of morbidity and
mortality in scleroderma, we next asked if our model would be useful
for the study of lung as well as skin fibrosis. Animals with Scl GVHD
(Fig. 6F), but not control animals (Fig. 6E),
show loss of the normal lacy alveolar pattern of lungs by day 21
post-BMT, with the decrease in alveolar space in Scl GVHD animals being
approximately 30% (Fig. 8). Lung
fibrosis has not been previously reported for this murine model. In the
inhibition experiment, Abs to TGF-ß prevented lung fibrosis as well
(Fig. 6G, plotted in Fig. 8). Anti-TGF-ß treatment had no
effect on the percentage of alveolar space in syngeneic BMT control
animals (Fig. 6H). The treatment of Scl GVHD and control
animals with nonspecific rabbit IgG also had no effect on skin or lung
fibrosis (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated that the Scl GVHD model faithfully reproduces
the skin fibrosis as well as the up-regulation of TGF-ß1 and collagen
mRNA synthesis that occurs in human scleroderma. Furthermore, we have
established the following temporal sequence of these events: TGF-ß1
mRNA up-regulation and CD11b+ mononuclear cell infiltration
are seen by day 7 post-BMT and precede detectable increases in collagen
mRNA and skin thickening (summarized in Table I, a composite of data
from several experiments).
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In contrast, the murine Scl GVHD model (B10.D2 > BALB/c) that we have characterized in the present study faithfully reproduces the most important features of human scleroderma, including skin thickening, mononuclear cell infiltrates, lung fibrosis, and up-regulation of cutaneous collagen and TGF-ß1 mRNA. Furthermore, the similarities between scleroderma and Scl GVHD, as well as the recent implication of microchimerism as a trigger for scleroderma, make the murine Scl GVHD model particularly appropriate for study. This model provides a system that can be manipulated to determine important variables in disease progression, and will allow us to test newly emerging therapeutic modalities at all stages of the disease, including the early stages when they may be more effective.
Central role of TGF-ß1 in Scl GVHD: proof of the hypothesis
mRNA for the cytokine TGF-ß1 is clearly up-regulated at early time points in animals with Scl GVHD. TGF-ß1, a potent stimulus for increased collagen synthesis, is thought to be critical in the cutaneous and pulmonary fibrosis of scleroderma (32, 33), as well as in other models of fibrosis (34, 35, 36, 37, 38). TGF-ß is made by multiple cell types, including monocytes, fibroblasts, and endothelial cells (39, 40), and can be converted to the active form by monocyte/macrophages (41). The three isoforms of human TGF-ß (TGF-ß1, TGF-ß2, and TGF-ß3) can be readily distinguished (42). These isoforms have pleuripotential effects on not only extracellular matrix homeostasis (32), but also on immune regulation (40, 43) and on epithelial growth (44). In human scleroderma, TGF-ß1 is thought to be the critical isoform implicated in cutaneous and pulmonary fibrosis (45, 46, 47).
We have shown that anti-TGF-ß Abs prevent the progression of skin and lung fibrosis in Scl GVHD at day 21, presumably by blocking TGF-ß; however, we have not tested the effects of the anti-TGF-ß Ab on other cytokines and the effect may be an indirect one. Because our Ab is polyclonal and inhibits all three isoforms of TGF-ß, we cannot make any conclusions about the role of the individual isoforms in murine Scl GVHD. The striking results of the Ab inhibition studies suggest the need for further study of these TGF-ß isoforms. Furthermore, anti-TGF-ß therapy with a humanized mAb may be useful in preventing disease progression in human Scl GVHD and scleroderma. In addition to anti-TGF-ß, experiments with Abs to other cytokines or to macrophage and T cell surface markers will be useful to further characterize the disease process in Scl GVHD and to identify other candidate molecules for immunotherapy.
Influence of genetic background on type, clinical manifestations, and effector cells of GVHD
Asai et al. (48) describe successful prevention of cytotoxic GVHD due to transplantation across major histocompatibility loci using blocking Abs to TGF-ß. The paradoxical results (TGF-ß produced by transplanted donor NK cells protected against GVHD in this model) point out the importance of the genetic backgrounds of donor and recipient individuals in such transplantations, and the diversity of GVHD-like reactions. Several quite different models of murine GVHD exist (reviewed in Refs. 49 and 50). They include transplantation from parental to nonirradiated F1 hybrid offspring (P > F1), in which autoimmune features like those in lupus erythematosus are commonly seen; transplantation across major histocompatibility loci, which is often rapidly lethal; and transplantation across minor histocompatibility loci, which most closely approximates in severity and course allogeneic sibling bone marrow transplantation in humans. In this last type of GVHD, somewhat different forms of disease can be produced experimentally in mice in carefully controlled depletion experiments by selecting for different cell types including the following: cytotoxic CD8 T cells, CD4 T cells, and NK cells (49, 51, 52, 53, 54). Therefore, the identity of effector cells may vary with the type of GVHD. Murine Scl GVHD is a subset of the last type of GVHD, exhibited by only a few transplantation pairs. It is not surprising, therefore, that different effector cells (presumably monocytes synthesizing TGF-ß1, rather than cytotoxic T cells attacking epithelium) operate in this distinctive fibrosing form of GVHD. The activation of host or donor monocytes by donor T cells is incompletely understood in this model, and will be an important parameter for examination in future experiments. Recently, Schlomchik et al. (55) determined that host APCs were required for the initiation of GVHD in a murine MHC-mismatch BMT model. Our model is also ideal for testing the involvement of host and donor APCs and T cells and the effects of chimerism in the development of Scl GVHD in an MHC-compatible BMT. Analysis of the diversity of murine GVHD-like reactions may be helpful in understanding the variability in type, development, and progression of human GVHD, and in understanding autoimmune disease itself.
In summary, we have shown that murine Scl GVHD faithfully reproduces the skin and lung fibrosis and up-regulation of cutaneous collagen and TGF-ß1 mRNA that occurs in human scleroderma. Cutaneous CD11b+ mononuclear cell infiltrates and increased TGF-ß1 and collagen mRNA precede dermal fibrosis and thickening, and the progression of early skin and lung disease can be inhibited with Abs to TGF-ß. Analysis of the diversity of murine GVHD-like reactions may be helpful in understanding the variability in type, development, and progression of human GVHD, and in understanding a complex autoimmune disease such as scleroderma. Most importantly, murine Scl GVHD is a useful model for testing potential interventions for scleroderma and GVHD.
Note. After submission of our manuscript it was reported that Abs to TGF-ß (the same R&D Ab (R&D Systems, Minneapolis, MN) used in our experiment) could reduce cutaneous sclerosis in a mouse model of bleomycin-induced scleroderma (56).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anita C. Gilliam, Department of Dermatology, Case Western Reserve University and University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address:
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; Scl GVHD, sclerodermatous GVHD; BMT, bone marrow transplantation; DC, dermal cells; PMN, polymorphonuclear neutrophils; E, experimental animals; C, control animals.
Received for publication May 19, 1999. Accepted for publication August 27, 1999.
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 A Crilly, J Hamilton, C J Clark, A Jardine, and R Madhok Analysis of transforming growth factor {beta}1 gene polymorphisms in patients with systemic sclerosis Ann Rheum Dis, August 1, 2002; 61(8): 678 - 681. [Abstract] [Full Text] [PDF]
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Y. Zhang, L. L. McCormick, S. R. Desai, C. Wu, and A. C. Gilliam Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma: Cutaneous Cytokines, Chemokines, and Immune Cell Activation J. Immunol., March 15, 2002; 168(6): 3088 - 3098. [Abstract] [Full Text] [PDF]
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 T. J. Gross and G. W. Hunninghake Idiopathic Pulmonary Fibrosis N. Engl. J. Med., August 16, 2001; 345(7): 517 - 525. [Full Text] [PDF]
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R. Abe, S. C. Donnelly, T. Peng, R. Bucala, and C. N. Metz Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites J. Immunol., June 15, 2001; 166(12): 7556 - 7562. [Abstract] [Full Text] [PDF]
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 O. EICKELBERG, A. PANSKY, E. KOEHLER, M. BIHL, M. TAMM, P. HILDEBRAND, A. P. PERRUCHOUD, M. KASHGARIAN, and M. ROTH Molecular mechanisms of TGF-{beta} antagonism by interferon {gamma} and cyclosporine A in lung fibroblasts FASEB J, March 1, 2001; 15(3): 797 - 806. [Abstract] [Full Text] [PDF]
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 K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Characterization of thyroid fibrosis in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., December 1, 2000; 68(6): 828 - 835. [Abstract] [Full Text]
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