This article is protected by copyright and is provided by Wisconsin TechSearch
`under license from Wolters Kluwer Health. All rights reserved.
`
`BASIC AND EXPERIMENTAL RESEARCH
`
`Effects of Pravastatin on Murine Chronic
`Graft-Versus-Host Disease
`
`Hyoung-Kyu Yoon,1 Ji-Young Lim,1 Tae-Jung Kim,2 Chul-Soo Cho,1 and Chang-Ki Min1,3
`
`Background. Chronic graft-versus-host disease (CGVHD) is a serious and increasingly common complication after
`allogeneic (allo) hematopoietic stem-cell transplantation, but currently available therapies have demonstrated limited
`efficacy. Furthermore, the statins have been reported to be effective in various immune-mediated disease models, but
`their therapeutic potentials versus CGVHD have not been determined.
`Methods. We used a B10.D23 BALB/c model of CGVHD, which differs at minor histocompatibility loci, to
`address the therapeutic effect of statins on the development of CGVHD. Pravastatin (PST, 30 mg/kg/day) was
`intraperitoneally injected for 5 days per week from the day of transplantation until 4 weeks after allo hematopoi-
`etic stem-cell transplantation.
`Results. The onset of clinical cutaneous GVHD was significantly slower in PST-treated recipients than in allo-controls
`(36 days vs. 25 days, respectively, P⬍0.05), and pathologic changes in skin disease confirmed this clinical result.
`Animals injected with PST showed less submucosal fibrosis in lungs than allo-controls. In addition, collagen deposition
`in skin and lungs was markedly attenuated by PST treatment. PST also significantly reduced protein concentrations and
`numbers of inflammatory and epithelial cells in bronchoalveolar lavage fluid. Significantly lower numbers of donor
`CD11b⫹ and CD4⫹, but not CD8⫹ cells, were observed in skin and bronchoalveolar lavage fluid after PST treatment.
`The protein concentrations of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell
`expressed and secreted (RANTES) in skin and lungs were substantially reduced in PST-treated animals when compared
`with allo-controls.
`Conclusions. This study suggests that the CGVHD-protecting effect of PST involves the down-regulation of chemo-
`kines and the reduction of collagen synthesis.
`
`Keywords: Chronic GVHD, Scleroderma, Pravastatin, Chemokines.
`
`(Transplantation 2010;90: 853–860)
`
`Allogeneic hematopoietic stem-cell transplantation (allo-
`
`HSCT) is an effective treatment for leukemia and genetic
`disorders. However, graft-versus-host disease (GVHD), op-
`portunistic infections, and the relapse of underlying disease
`are major obstacles. Chronic GVHD (CGVHD) is an increas-
`ingly common complication of allo-HSCT, and its incidence
`has been reported to be as high as 80% in some series. Fur-
`thermore, its incidence is believed to be increasing, at least in
`
`This work was supported by Catholic Medical Center Research Foundation.
`1 Department of Internal Medicine, The Catholic University of Korea, Seoul,
`Korea.
`2 Department of Hospital Pathology, The Catholic University of Korea,
`Seoul, Korea.
`3 Address correspondence to: Chang-Ki Min, M.D., Department of Internal
`Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea,
`505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea.
`E-mail: ckmin@catholic.ac.kr
`H.-K.Y. participated in research design and writing of the manuscript; J.-Y.L.
`participated in performance of the research; T.-J.K. participated in per-
`formance of the research (histopathology); C.-S.C. participated in data
`analysis; and C.-K.M. participated in research design and writing of the
`manuscript.
`Received 19 October 2009. Revision requested 7 November 2009.
`Accepted 19 July 2010.
`Copyright © 2010 by Lippincott Williams & Wilkins
`ISSN 0041-1337/10/9008-853
`DOI: 10.1097/TP.0b013e3181f2c92b
`
`part, because of the use of peripheral blood as a stem-cell
`source, nonmyeloablative conditioning, withdrawal of im-
`munosuppression to induce antitumor activity, longer sur-
`vival because of better supportive care, and increases in the
`numbers of patients receiving donor leukocyte infusions
`(1, 2). The treatment of CGVHD remains a formidable chal-
`lenge. Even with a wide range of therapeutic options,
`CGVHD and infectious complications that arise during its
`management are major causes of late mortality among allo-
`HSCT recipients (3). Furthermore, the model systems devel-
`oped to examine CGVHD are limited. Of these, the B10.D2
`(H-2d)3 BALB/c (H-2d), major histocompatibility complex-
`matched and minor histocompatibility antigen–mismatched
`model of GVHD shows characteristics resembling human
`CGVHD, such as a relatively late onset, skin fibrosis, alopecia,
`and lung fibrosis with increased collagen deposition (4 – 6).
`The statins (3-hydroxy-3-methyl coenzyme reductase
`inhibitors) are a class of cholesterol-lowering drugs that re-
`duce mortality resulting from cardiovascular disease and
`stroke, but they also have strong immunomodulatory effects
`on antigen-presenting cells (APCs) and T cells and interfere
`with the synthesis of L-mevalonate and its downstream iso-
`prenoid metabolites (7). It has been demonstrated that some
`statins inhibit the productions of proinflammatory cytokines
`
`Transplantation • Volume 90, Number 8, October 27, 2010
`
`www.transplantjournal.com | 853
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`854 | www.transplantjournal.com
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`Transplantation • Volume 90, Number 8, October 27, 2010
`
`and that they are beneficial in patients with chronic inflam-
`matory disorders (8, 9). Recently, several studies on GVHD
`and statins have been performed using murine models of
`acute GVHD (AGVHD) with donor and recipient strains
`fully mismatched at major histocompatibility complex and
`minor histocompatibility loci (10). However, much remains
`unknown about the pathophysiology of CGVHD, and al-
`though AGVHD and CGVHD are similar in some respects, it
`seems likely that the two diseases have different initiation
`requirements and pathogenic mechanisms. More impor-
`tantly from the experimental view, it is not obvious whether
`findings based on the AGVHD models apply to CGVHD syn-
`drome (11). Moreover, few experimental studies have been
`conducted using CGVHD and statins, and thus, their molec-
`ular modes of action against the alloimmune reactions under-
`lying CGVHD have not been clarified fully. In this study, we
`used a murine CGVHD model, which shares critical patho-
`logic features with human CGVHD, to investigate the effects
`of pravastatin (PST) on the occurrence of CGVHD.
`
`MATERIALS AND METHODS
`Mice and Experimental Allo-HSCT
`Female B10.D2 (H-2d) and BALB/c (H-2d) mice (8- to 12-week old) were
`purchased from Japan Shizuoka Institute for Laboratory Animals (Japan
`SLC, Shizuoka, Japan). B10.D2 and BALB/c mice were used as donors and
`recipients, respectively, for allo-HSCT to produce CGVHD (5). Briefly, re-
`cipient mice were lethally irradiated with 700 cGy using a Gammacel 137Cs
`source. Approximately 6 hr later, they were injected intravenously through a
`tail vein with donor spleen (1.5⫻106/mouse) and bone marrow (BM) cells
`(3⫻106/mouse) suspended in Roswell Park Memorial Institute 1640 me-
`dium (WelGENE Inc., Daegu, South Korea) containing 10 U/mL heparin
`(Fisher Scientific, Pittsburgh, PA; referred to as the allo-control). PST was
`administered as described later after allo-HSCT (referred to as the allo-PST).
`In addition, radiated BALB/c recipient mice also received BALB/c spleen and
`BM cells (syngeneic [syn] HSCT, referred to as the syn-control). The dose of
`donor cells used in these experiments was determined using literature values
`(5) and by performing pilot experiments (data not shown). Degrees of clin-
`ical GVHD were assessed every 3 days based on the occurrences of skin
`lesions, such as alopecia and dermatitis.
`
`PST Preparation and Treatment
`PST (Merck, Darmstadt, Germany) was prepared as a 4 mg/mL stock
`solution. Briefly, 4 mg of PST was dissolved in 100 ␮L of saline, and the total
`volume of this solution was adjusted to 1 mL. PST (30 mg/kg/100 ␮L) was
`administered intraperitoneally (IP) for 5 consecutive days per week (from
`day 1 to 28). These injection conditions were chosen based on the findings of
`a pilot study (data not shown).
`
`Bronchoalveolar Lavage
`Bronchoalveolar lavage (BAL) was performed in situ four times with
`Hanks’ balanced salt solution (35 mL/kg; pH 7.2–7.4, WelGENE Inc.), and
`total protein was determined in supernatant using the Bradford method (12),
`as recommended by the manufacturer (Bio-Rad Laboratories, Hercules,
`CA). Pooled cell pellets were combined and resuspended in 1 mL of Hanks’
`balanced salt solution, and numbers of BAL cells was counted using a hemo-
`cytometer. Aliquots (100 ␮L) of cell suspensions were cytocentrifuged, and
`cytospin slides were stained with Diff-Quick for differential cell counting.
`
`Preparation of Skin Cell Suspension
`Small pieces of depilated skin were digested in Roswell Park Memorial
`Institute medium-10% fetal bovine serum (WelGENE Inc) containing
`0.27% collagenase (Sigma-Aldrich, St. Louis, MO), 0.01% DNase (Sigma),
`and 1000 U/mL hyaluronidase (Worthington Biochemical Corp, Lakewood,
`
`NJ) at 37°C for 2 hr. The digested skin was filtered through 70-␮m cell
`strainer Falcon (Bectin Dickenson Labware, Franklin, Lakes, NJ) to generate
`single-cell suspensions.
`
`Histologic Analysis and Immunostaining
`Formalin-fixed, paraffin-embedded tissue sections were hematoxylin-
`eosin (H&E) stained. Frozen lung and skin sections were immediately embed-
`ded in OCT embedding medium (Sakura, Ted Pella, Redding, PA) and
`cryosectioned for immunostaining. For collagen I staining, cryosections were
`first incubated with rabbit anti-mouse collagen I at a dilution of 1:200 (Chemi-
`con International, Temecula, CA) and then with Alexa Fluor 488-conjugated
`donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA). After extensive washing,
`samples were counterstained with 4,6-diamidino-2-phenylindole (Sigma)
`and embedded in Vectashield (Vector Laboratories, Burlingame, CA). De-
`grees of CGVHD in skin were evaluated by measuring dermal thicknesses at
`the dermo-epidermal and dermis-subcutaneous fat junctions under a mi-
`croscope. The presence of CGVHD in lungs was evaluated by assessing
`chronic airway rejection and obliterative bronchiolitis, as determined by
`Stewart et al. (13).
`
`Flow Cytometry
`surface phenotypes, cells were stained with
`To determine cell
`phycoerythrin-conjugated monoclonal antibodies (MoAbs) to CD4, CD8,
`and CD11b. All MoAbs were purchased from BD Pharmingen (San Diego,
`CA). Samples collected in heparin (50 ␮L) were incubated on ice for 30 min
`with mouse anti-mouse MoAbs and washed with phosphate-buffered saline
`(PBS; WelGENE Inc.) containing 0.05% sodium azide and fixed with
`PBS/1% paraformaldehyde. All samples were analyzed using a FACScan cy-
`tometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).
`
`Protein Extraction and Measurements of
`Chemokines by ELISA and Western Blotting
`Tissue samples were homogenized in 2 mL buffer solution (1⫻ PBS, 1%
`Nonidet P-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate, and 1
`tablet of Complete Protease Inhibitor Cocktail [Roche Diagnostics, Basel,
`Switzerland]), centrifuged at 3000 rpm for 20 min, and supernatants were
`harvested. Total protein concentrations in supernatant were determined us-
`ing Bio-Rad Protein Assays (Bio-Rad Laboratories). Concentrations of
`regulated on activation normal T cell expressed and secreted (RANTES) and
`monocyte chemoattractant protein-1 (MCP-1) (R&D Systems, Minneapolis,
`MN) were measured in supernatants of homogenized tissues. Assays were
`performed according to the manufacturer’s protocol. Assay sensitivities were
`less than 2.0 pg/mL for RANTES and less than 9 pg/mL for MCP-1. ELISA
`plates were read using a microplate reader (Bio-Rad Laboratories). Protein
`sample from cell lysates, 40 ␮g, was separated using sodium dodecyl sulfate
`polyacrylamide gel electrophoresis. Proteins were then transferred to nitro-
`cellulose membranes and immunoblotted. MCP-1 antibody was purchased
`from Cell Signaling Technology (Beverly, MA), RANTES antibody from Ab-
`cam Inc. (Cambridge, MA), and donkey anti-rabbit IgG antibody from Santa
`Cruz Biotechnology (Santa Cruz, CA).
`
`Statistical Analysis
`All values are expressed as means⫾SEMs. Statistical comparisons between
`groups were performed using the parametric independent sample t test if
`there were more than or equal to five animals per group or using the Mann-
`Whitney U test if there were less than five animals per group. Wilcoxon’s rank
`test was used to analyze survival data. P values less than 0.05 were considered
`significant.
`
`RESULTS
`PST Attenuated Skin and Pulmonary Pathologic
`Lesions
`Because there is growing evidence that statins have im-
`munomodulatory activities, we asked whether PST could
`
`

`

`© 2010 Lippincott Williams & Wilkins
`
`Yoon et al.
`
`855
`
`prevent the development of CGVHD of lung and skin. PST
`(30 mg/kg) was administered IP for 5 consecutive days per
`week (from day 1 to 28) after transplantation. Lethally irradi-
`ated BALB/c mice were transplanted by tail vein injection
`with BM and spleen cells from B10.D2 mice and developed
`skin lesions with alopecia or scaling on ears, tails, and paws.
`Control BALB/c animals receiving syn transplants did not
`develop skin lesions. In vivo treatment with PST suppressed
`the incidence and severity of clinical skin CGVHD when
`compared with allo-controls injected with diluent. The onset
`of clinical cutaneous GVHD was significantly slower and less
`severe in the animals treated with PST than in the allo-con-
`trols (Fig. 1A, 36 days vs. 25 days, 80% vs. 50%, respectively,
`P⬍0.05). After day 60, all allo-recipients clinically bounced
`back from CGVHD regardless of PST treatment. Consistent
`with clinical results, histologic examinations revealed thick-
`
`ening of the epithelial layer, loss of hair follicles and of sub-
`dermal fat, ulcers in epithelial and dermal layers, and heavy
`collagen deposition in the skin lesions of allo-controls. As
`shown in Figure 1(B), thickening of dermis in allo-controls
`was markedly increased compared with syn-controls. In stark
`contrast, allo-PST mice had much less severe lesions accord-
`ing to the pathologic categories mentioned earlier. All allo-
`controls exhibited the pathologic changes regardless of the
`presence of clinical cutaneous lesions. Findings of routine
`histopathologic sections showed that there was 30% increase
`in the dermal thickness in allo-controls compared with syn-
`controls. PST treatment significantly reduced dermal thick-
`ening compared with allo-controls at day 14; the increase of
`dermal thickness was just 18% compared with syn-controls
`(Fig. 1C). Dermal thickenings at 28 and 56 days in the allo-
`
`B
`
`}
`
`A
`
`100
`
`:: 75
`.,
`.::
`ti 50
`.,
`:::
`tll
`C 25
`::::,
`
`syn-control
`-
`allo-control
`-
`-+- allo-PST
`
`0
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`C
`
`45
`
`■ allo-control
`Il alio-PST
`
`D
`
`D14
`
`D28
`
`D56
`
`syn-
`control
`
`allo-
`control
`
`allo-
`PST
`
`syn-
`control
`
`allo-
`control
`
`allo-
`PST
`
`C
`
`.. C.
`
`40
`"'
`.. ~
`·co 35
`ti C
`=i 25
`0
`·-
`~~
`c-- ,:,
`"' 0
`20
`....
`: e 15
`t; 5 10
`.5 0
`....
`
`30
`
`5
`
`0
`
`D28
`D14
`D56
`D28
`D14
`D56
`FIGURE 1. Pravastatin (PST) treatment ameliorated the severity of cutaneous and pulmonary chronic graft-versus-host
`disease (CGVHD). After being administered a lethal dose of radiation (700 cGy), BALB/c mice were transplanted with
`B10.D2 donor spleen (1.5⫻106/mouse) and bone marrow cells (3⫻106/mouse) as described in Materials and Methods. These
`mice were then administered (intraperitoneally) phosphate-buffered saline (the allogeneic [allo]-control, n⫽10) or PST (30
`mg/kg, allo-PST, n⫽10) for 5 consecutive days per week (from day 1–28) post-hematopoietic stem-cell transplantation
`(HSCT). A control group of BALB/c recipient mice received BALB/c spleen and bone marrow cells (syngeneic [syn]-control,
`n⫽6). (A) The occurrence of skin CGVHD was monitored for 60 days after transplantation. Data obtained from two experi-
`ments were combined. *P less than 0.05 between the allo-control and allo-PST groups. (B) Representative histology of skin
`at days 14, 28, and 56 post-HSCT. Mice with CGVHD had thick skins with influx of infiltrates of mononuclear cells from day
`14 post-HSCT. Syn-controls (BALB/c4BALB/c) showed no change in skin thickness or inflammation. PST markedly reduced
`skin thickenings and inflammatory cell infiltrations caused by CGVHD. (C) Pathology scores of skin for the representative
`mice. Bars represent means⫾SEMs of percentage increases in dermal thicknesses of the allo-PST (n⫽5) and allo-control
`groups (n⫽5), respectively, compared with those of the syn-control group (n⫽3). The paired t test revealed a significant
`difference between the allo-control and the allo-PST groups at days 14, 28, and 56. *P less than 0.05; **P less than 0.01.
`Dermal thicknesses were determined using hematoxylin-eosin (H&E)-stained tissue sections by image analysis, as de-
`scribed in Materials and Methods. (D) Histopathology of lungs in the syn-control, the allo-control, and the allo-PST groups
`was examined on days 14, 28, and 56 post-HSCT. H&E staining; magnification, ⫻400. Allo-controls had more severe sub-
`mucosal fibrosis than syn-controls. PST treatment markedly reduced fibrosis on day 56. However, fibrosis was observed in
`the allo-PST group on day 28, although it was less than that observed in the allo-controls. Syn-control mice did not develop
`submucosal fibrosis as determined by histology.
`
`

`

`856 | www.transplantjournal.com
`
`Transplantation • Volume 90, Number 8, October 27, 2010
`
`A skin
`syn-control
`
`allo-control
`
`allo-PST
`
`B
`
`lung
`syn-control
`
`allo-control
`
`allo-PST
`
`FIGURE 2. Pravastatin (PST)
`treatment
`prevented the deposition of type I collagen
`in skin and lungs. Tissue sections of skin (A)
`and lungs (B) from syngeneic (syn)-control,
`allogeneic (allo)-control, and allo-PST mice
`were stained with type 1 collagen. Images
`were acquired using an Olympus C-3000
`zoom camera and processed using Image-
`Pro Plus, version 4 (Media Cybernetics, Inc.,
`Bethesda, MD). Magnification, ⫻200.
`
`PST mice were 20% and 13%, respectively, greater than those
`in syn-controls.
`Because pulmonary fibrosis is a major cause of morbid-
`ity and mortality in CGVHD, we next asked whether our
`model would be useful for the study of lung and skin fibrosis.
`In the lungs, airway submucosal fibrosis was observed on days
`28 and 56 in allo-controls. PST injection markedly reduced
`fibrosis on day 56 although some fibrosis was observed on day
`28. Syn-controls did not develop GVHD clinically or have
`submucosal fibrosis by histologic examinations (Fig. 1D).
`
`PST Treatment Inhibited Type 1 Collagen
`Deposition
`Fibrosis, regardless of its cause, is characterized by ex-
`tracellular matrix deposition of predominantly collagen type
`I (14). The progressive accumulation of connective tissue re-
`sults in the destruction of the normal tissue architecture and
`internal organ failure. In CGVHD, the severity of skin and
`internal organ fibrosis is correlated with clinical disease
`course (15). Thus, we stained skin and lung tissues with ant-
`icollagen type I at day 28 after HSCT. As shown in Figure
`2(A), collagen deposition was greater in the dermis of allo-
`controls compared with syn-controls, whereas dermal colla-
`gen deposition was markedly reduced by PST treatment. In
`the lungs, excessive lung collagen deposition was also noted in
`the interstitium of allo-controls when compared with syn-
`controls; pulmonary collagen deposition was also signifi-
`cantly diminished by PST treatment (Fig. 2B).
`
`PST Reduced Skin and Lung Inflammation
`We next evaluated the effects of PST on early cutaneous
`and pulmonary inflammation induced by CGVHD. PST-
`treated allo-recipients exhibited marked reduction in mean
`skin total cell counts versus allo-controls at days 14 and 28
`after transplantation (Fig. 3A, P⬍0.01). In lungs, PST treat-
`
`□ syn-control
`■ allo-control
`
`■ allo-PST
`
`D14
`
`D28
`
`A Jo
`25
`
`~
`0
`'";;
`
`20
`
`15
`
`10
`
`5
`
`0
`
`B
`
`500
`450
`400
`..J 350
`E 300
`~ 250
`200
`150
`100
`50
`0
`
`D14
`D28
`FIGURE 3. Effect of pravastatin (PST) on skin cell numbers
`(A) and protein concentrations of bronchoalveolar lavage
`(BAL) fluid (B). (A) Lethally irradiated BALB/c mice were
`transplanted with B10.D2 or BALB/c spleen and bone marrow
`cells, and skin cell suspensions were prepared, as described
`in Materials and Methods. The absolute numbers of mononu-
`clear cells in skin cell suspensions were determined at each
`time point. Open bars, syngeneic (syn)-controls (n⫽3); closed
`bars, allogeneic (allo)-controls (n⫽5); and gray bars, allo-PST
`mice (n⫽5). Data are expressed as the means⫾SEM; **P less
`than 0.01. Data from one of two similar experiments are
`shown. (B) BAL fluid protein concentrations were analyzed
`using the Bradford method. Open bars, syn-controls; closed
`bars, allo-controls; gray bars, allo-PST mice. Data are ex-
`pressed as means⫾SEM; n⫽5 animals per group. *P less than
`0.05.
`
`

`

`© 2010 Lippincott Williams & Wilkins
`
`Yoon et al.
`
`857
`
`TABLE 1.
`
`Group
`
`Syngeneic
`control
`Allogeneic
`control
`Allogeneic
`PST
`
`Reduction in the severity of lung inflammation by PST treatment
`Macrophages (ⴛ105)
`Lymphocytes (ⴛ105) Neutrophils (ⴛ105)
`Total cells (ⴛ105)
`D14
`D28
`D14
`D28
`D14
`D28
`D14
`D28
`
`Epitherial cells (ⴛ105)
`D14
`D28
`
`17.5 ⫾ 1.6
`
`29.4 ⫾ 4.2
`
`13.7 ⫾ 1.4
`
`21.3 ⫾ 3.7
`
`0.2 ⫾ 0.0
`
`0.8 ⫾ 0.3
`
`0.1 ⫾ 0.0
`
`0.3 ⫾ 0.2
`
`3.5 ⫾ 0.4
`
`.0 ⫾ 1.1
`
`48.3 ⫾ 5.0
`
`82.0 ⫾ 8.2
`
`19.2 ⫾ 5.1
`
`54.9 ⫾ 2.5
`
`6.7 ⫾ 0.9
`
`9.8 ⫾ 3.3
`
`0.4 ⫾ 0.2
`
`0.4 ⫾ 0.2 22.0 ⫾ 2.4
`
`17.5 ⫾ 3.0
`
`26.2 ⫾ 1.7a 42.0 ⫾ 6.0a 15.6 ⫾ 1.7
`
`32.4 ⫾ 4.9a
`
`2.3 ⫾ 0.5a 2.2 ⫾ 0.5b 0.3 ⫾ 0.1
`
`0.4 ⫾ 0.2
`
`9.5 ⫾ 2.4a
`
`7.1 ⫾ 1.0a
`
`Data are the mean⫾SEM (n⫽6).
`a P⬍0.01 compared with the allogeneic control group.
`b P⬍0.05.
`PST, pravastatin.
`
`skin
`CD11b
`
`A
`
`8
`7
`6
`
`~
`4
`M 3
`2
`
`1.0
`
`0.8
`
`0.6
`'&
`□ syn-control ~ 0.4
`■ allo-control
`
`0.2
`
`CD4
`
`CDS
`
`1.0
`
`0.6
`
`0.4
`
`0
`□ syn-control ~
`
`■ allo-control
`
`□ syn-control
`
`■ allo-control
`
`FIGURE 4. Effect of pravastatin
`(PST) on numbers of CD11b⫹,
`CD4⫹, and CD8⫹ T cells in target
`organs. Cell numbers in skin (A)
`and bronchoalveolar lavage fluid
`(B) at days 14 and 28 posthemato-
`poietic stem-cell transplantation
`were counted and phenotyped by
`flow cytometry, as described in
`Materials and Methods. Open bars,
`syngeneic (syn)-controls; closed
`bars, allogeneic (allo)-controls;
`and gray bars, allo-PST mice. The
`results are representative of two
`independent series of experi-
`ments. Results are means⫾SEMs;
`n⫽3 to 4 animals per group,**P
`less than 0.01. BMT, bone marrow
`transplantation.
`
`■ allo-PST
`
`0.0
`
`■ allo-PST
`
`D14
`
`D28
`
`Days after BMT
`
`CD4
`
`B
`
`25
`
`20
`~ 15
`M 10
`
`5
`
`0
`
`D14
`
`D28
`
`Days after BMT
`
`lung
`CD11b
`
`D14
`
`D28
`
`Days after BMT
`
`25
`
`20
`
`0 15
`□ syn-control M 10
`■ allo-control
`
`■ allo-PST
`
`0
`
`20
`
`0
`
`15
`□ syn-control M 10
`■ allo-control
`5
`
`■ allo-PST
`
`D14
`
`D28
`
`Days after BMT
`
`0
`
`D14
`
`D28
`
`Days after BNT
`
`0.2
`
`0.0
`
`D14
`
`D28
`
`Days after BMT
`
`CDS
`
`0.8 L
`25 L
`
`■ allo-PST
`
`□ syn-control
`
`■ allo-control
`
`■ allo-PST
`
`ment significantly decreased total cell numbers in BAL fluid
`at all times when compared with allo-controls (Table 1).
`Moreover, PST treatment markedly reduced protein concen-
`trations in BAL fluid (a marker of lung permeability) on days
`14 and 28 (Fig. 3B, P⬍0.05). We next performed a micro-
`scopic examination of BAL fluid because BAL fluid cellularity
`can be used as a surrogate marker of lung inflammation
`caused by CGVHD (16) and performed differential counts of
`macrophages, lymphocytes, and neutrophils using morpho-
`logic criteria. As shown in Table 1, numbers of lymphocytes
`were significantly attenuated in the allo-PST group from day
`14. Furthermore, alveolar macrophages and lymphocytes
`were significantly reduced in allo-PST animals at day 28.
`Neutrophil counts in BAL fluid were not different between
`the two groups, but the numbers of epithelial cells, which
`reflect degree of alveolar injury, were significantly lower at
`days 14 and 28 in the allo-PST group. These results indicate
`that PST treatment significantly attenuated skin and pulmo-
`nary inflammation caused by CGVHD.
`It has been noted that initial target organ inflammation
`is caused primarily by CD11b⫹ monocytes and T cells (17,
`18). We performed flow cytometric analysis on cells in skin
`cell suspensions and BAL fluid using CD4, CD8, and CD11b
`surface markers. After allo-HSCT, the numbers of CD11b⫹
`
`and CD4⫹ T cells, but not CD8⫹ cells, were markedly in-
`creased in skin and BAL fluid, and PST administration inhib-
`ited these increased infiltrations (Fig. 4A,B, respectively).
`
`MCP-1 and RANTES Expression in Target
`Tissues Was Reduced by PST Administration
`MCP-1 and RANTES have been shown to play the im-
`portant roles by chemoattracting immunocompetent cells
`during pathogenesis of this CGVHD model (5, 6). So, we
`hypothesized that the reduction in CGVHD caused by PST
`treatment would be because of the inhibition of chemokine
`expression, such as MCP-1 and RANTES. Accordingly, we
`measured the protein levels of MCP-1 and RANTES by ELISA
`in the skin and lung tissues to determine whether PST affects
`chemoattraction of these monocytes to the target tissues by
`influencing their expressions.
`Although the concentrations of MCP-1 and RANTES
`were higher in allo-controls on days 14 and 28 compared with
`syn-controls, they were significantly reduced by PST treat-
`ment on day 14 but not on day 28. (Fig. 5A,B). In particular,
`the protein levels of RANTES in skin on day 28 were signifi-
`cantly reduced by PST treatment. Western blot analysis
`showed again that PST markedly reduced the chemokine ex-
`
`

`

`858 | www.transplantjournal.com
`
`Transplantation • Volume 90, Number 8, October 27, 2010
`
`C skin MCP-1 RANTES
`
`allo-
`allo-
`control PST
`
`allo-
`control
`
`- · 11-
`
`II
`
`allo-
`PST
`
`~
`
`B-actin
`
`D syn-rontol
`■ allo-conlrol
`■ alkH'ST
`
`0
`
`0.8
`0.7
`0.6
`0.5
`0.4
`~ 0.3
`0.2
`0.1
`0
`
`MCP-1
`
`RANTES
`
`A
`
`skin
`MCP-1
`
`RANTES
`
`140
`120
`,:100
`.J .Sl
`E e 80
`l~ 60
`S 40
`20
`0
`
`120
`100
`80
`60
`40
`20
`0
`
`D14
`D28
`Days after BMT
`
`D14
`D28
`Days after BMT
`
`B lung
`MCP-1
`
`RANTES
`
`50
`
`40
`
`C
`.J $ 30
`E e
`i~20
`~ 10
`
`0
`
`350
`300
`250
`200
`150
`100
`50
`0
`
`D14
`D28
`Days after BMT
`
`D28
`D14
`Days after BMT
`
`■ allo-control
`■ allo-PST
`
`B-actin
`
`■ allo-control
`
`■ allo-PST
`
`allo-
`allo-
`control PST
`
`allo-
`allo-
`control PST
`
`D lung MCP-1 RANTES
`1-- 11-
`1--11
`.
`
`D Syn-<:OOlrol
`■ alkH:onlrol
`■ allo-PST
`
`0.7
`0
`.:: 0.6
`!'!!
`0.5
`0.4
`0.3
`0.2
`0.1
`0.0
`
`.
`I
`
`0.8 J
`
`MCP-1
`RANTES
`FIGURE 5. The protein levels of MCP-1 and RANTES in the target tissues at 14 and 28 days posthematopoietic stem-cell
`transplantation were substantially reduced by pravastatin. Lethally irradiated BALB/c mice were transplanted with B10.D2
`or BALB/c spleen and bone marrow cells and tissue extracts were prepared, as described in Materials and Methods. The
`protein concentrations of MCP-1 and RNATES were measured by ELISA in skin (A) and lung (B) tissue protein extracts.
`Results are means⫾SEMs; n⫽3 to 4 animals per group, *P less than 0.05, **P less than 0.01. Western blotting was also
`performed using skin (C) and lung (D) tissue protein extracts from allogeneic (allo) recipients. Results are expressed as
`means⫾SEMs; n⫽3 to 4 animals per group, *P less than 0.05. syn, syngeneic.
`
`pressions in the protein extracts from the skin and lungs (Fig.
`5C,D). These data demonstrate that PST reduces monocytes
`and T-cell infiltration into skin and lungs by attenuating the
`expressions of MCP-1 and RANTES early after transplant.
`
`DISCUSSION
`Protective effects of statins in different cancer models
`(19) and in allograft rejection and autoimmunity models (8,
`20) have been established by pleiotropic mechanisms. Re-
`cently, a report was issued on the treatment of experimental
`AGVHD with atorvastatin (10). Therefore, we investigated
`whether PST treatment affects the development of CGVHD
`in a murine CGVHD model, which shares critical character-
`istics with human CGVHD. Our results indicate that PST
`significantly reduces inflammation and collagen deposition
`in CGVHD target organs, such as skin and lungs, by effec-
`tively blocking the influx of effector cells by down-regulating
`the expressions of MCP-1 and RANTES.
`Recent studies in mice (10) and humans (21) suggest
`that harnessing the immunomodulatory properties of statins
`(cholesterol-lowering drugs that are prescribed worldwide to
`prevent and treat atherosclerosis) might be a more effective
`and less toxic way of controlling GVHD. The therapeutic ef-
`fects of statins on cardiovascular and autoimmune disease
`
`seem to be because of their antiinflammatory effect, which is
`also relevant in CGVHD, because it has an inflammatory
`component (22). However, relatively few studies have been
`conducted in vivo on CGVHD, and thus, the precise mecha-
`nisms of the actions of statins against CGVHD have not been
`elucidated. CGVHD is often diagnosed based on its distinct
`clinicopathologic features regardless of time of onset (23).
`AGVHD typically presents with inflammatory skin, gastroin-
`testinal, and hepatic disease, whereas CGVHD is character-
`ized by cutaneous fibrosis, myositis, hepatic disease, and the
`involvement of exocrine glands. The fibrosing variant of
`CGVHD, sclerodermatous (scl) CGVHD, accounts for ap-
`proximately 10% to 15% of CGVHD cases (24) and is less well
`understood than the AGVHD. Like scleroderma, individuals
`with scl GVHD develop skin and lung fibrosis, and the disease
`is typically refractory to immunosuppressant treatment. A
`murine allo-HSCT model generated by transplanting B10.D2
`BM and spleen cells into lethally irradiated BALB/c mice has
`been established to probe the mechanisms that initiate scl
`GVHD. In these studies, BALB/c mice transplanted with syn
`BALB/c BM and spleen cells served as non-CGVHD controls,
`and it was found that many donor cells are present in the skins
`of mice with CGVHD but not in syn BM-transplanted con-
`trols (5, 6). Its dominant features include skin fibrosis result-
`
`

`

`© 2010 Lippincott Williams & Wilkins
`
`Yoon et al.
`
`859
`
`ing from increased collagen deposition, follicular dropout,
`loss of subdermal fat, and dermal mononuclear infiltrates. In
`addition, pulmonary fibrosis has also been observed in this
`murine model (5) as have inflammation and the destructions
`of salivary and lacrimal glands. These clinical features were
`also observed in allo-controls in our experiments.
`During the characterization of this CGVHD model, ini-
`tial inflammation in target organs was attributed primarily to
`CD11b⫹ mononuclear cells and T cells. Especially CD4⫹ T
`cells have also been reported to be activated in spleens during
`early murine CGVHD (25). In line with other reports, subsets
`of infiltrating T cells in skin and lungs were identified among
`CD4⫹ but not among CD8⫹ T cells. PST-treated animals
`showed markedly less CD4⫹ T-cell infiltration when com-
`pared with allo-controls, but no significant difference in
`extents of CD8⫹ T-cell infiltration was observed between
`allo-PST and allo-control groups. Because the earliest initiat-
`ing events of self-reactivity would be expected to occur
`through APCs-T cell interactions in spleen in transplanted
`recipients (26), we also examined the expansion of splenic
`cellularity after transplantation. In contrast to the marked
`reductions in CD11b⫹ mononuclear cell and CD4⫹ T-cell
`infiltration in skin and lungs achieved by PST treatment, in
`spleen, the numbers of CD11b⫹ cells or CD4⫹ and CD8⫹ T
`cells were not different between allo-PST and allo-control
`groups (data not shown), indicating that PST did influence
`effector cell migration into the target organs rather than the ex-
`pansion of donor CD11b⫹ or T cells. Effector cells in GVHD
`leave the general circulation and migrate into tissues through a
`well-ordered series of events (27), and chemokines and their re-
`ceptors are important mediators of this trafficking.
`In this study, we examined the effect of PST on
`CGVHD to elucidate its mode of action, by focusing on its
`effect on effector cell trafficking. The migration and recruit-
`ment of leukocytes to specific tissues is a multistep process
`that involves the sequential activations of various adhesion
`molecules on immune cells and vascular endothelium and the
`activations of a vast array of chemokines (28, 29). Chemo-
`kines and their receptors have been implicated in the patho-
`genesis of scleroderma (30), and by recruiting immune cells
`to target tissues, they are essential contributors to tissue dam-
`age. In this CGVHD model, high levels of chemokine
`mRNAs, namely CCL2 (MCP-1), CCL5 (RANTES), CCL17,
`and interferon-␥ inducible
`chemokines
`(CXCL9/Mig,
`CXCL10/interferon gamma-induced protein 10 kDA
`(IP-10), and CXCL11/I-TAC), which are all monocyte or
`macrophage and T-cell