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Fibrosis by Serum Amyloid PReduction of Bleomycin-Induced Pulmonary

Richard H. GomerRonkainen, Jeff R. Crawford, Elizabeth L. Travis and Darrell Pilling, David Roife, Min Wang, Sanna D.

http://www.jimmunol.org/content/179/6/4035doi: 10.4049/jimmunol.179.6.4035

2007; 179:4035-4044; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Reduction of Bleomycin-Induced Pulmonary Fibrosis by SerumAmyloid P1

Darrell Pilling,2* David Roife,* Min Wang,† Sanna D. Ronkainen,* Jeff R. Crawford,*Elizabeth L. Travis,† and Richard H. Gomer*

Fibrotic diseases such as scleroderma, severe chronic asthma, pulmonary fibrosis, and cardiac fibrosis kill tens of thousands ofpeople each year in the U.S. alone. Growing evidence suggests that in fibrotic lesions, a subset of blood monocytes enters the tissueand differentiates into fibroblast-like cells called fibrocytes, causing tissue dysfunction. We previously found that a plasma proteincalled serum amyloid P (SAP) inhibits fibrocyte differentiation in vitro. Bleomycin treatment is a standard model for pulmonaryfibrosis, and causes an increase in collagen, fibrocytes, and leukocytes in the lungs, and a decrease in peripheral blood hemoglobinoxygen saturation. We find that injections of rat SAP in rats reduce all of the above bleomycin-induced changes, suggesting thatthe SAP injections reduced the bleomycin-induced pulmonary fibrosis. We repeated these studies in mice, and find that injectionsof murine SAP decrease bleomycin-induced pulmonary fibrosis. To confirm the efficacy of SAP treatment, we used a delayedtreatment protocol using SAP from day 7 to 13 only, and then measured fibrosis at day 21. Delayed SAP injections also reducethe bleomycin-induced decrease in peripheral blood hemoglobin oxygen saturation, and an increase in lung collagen, leukocyteinfiltration, and fibrosis. Our data suggest the possibility that SAP may be useful as a therapy for pulmonary fibrosis inhumans. The Journal of Immunology, 2007, 179: 4035–4044.

P ulmonary fibrosis is a common problem of end-stage lungdisease caused by environmental toxins, radiation, or che-motherapy treatments for cancer or many chronic inflam-

matory diseases (1–3). Fibrosis leads to reduced lung function andhas a high mortality rate (4). The processes that drive fibrosis aredynamic, involving infiltrating leukocytes, activation and prolifer-ation of fibroblast-like cells, destruction of the alveolar structures,and the deposition of extracellular matrix proteins. These eventsappear to be due to repeated and/or aberrant repair events (1, 5).There is currently no Food and Drug Administration-approvedtherapy for pulmonary fibrosis or other fibrosing diseases (6).

Bleomycin is the primary chemotherapeutic drug for testicularcancer, but lung fibrosis is a side effect in �10% of patients (2).Bleomycin-induced lung fibrosis is the most frequently used ro-dent model of lung fibrosis, and produces inflammatory and fi-brotic events similar to those seen in human pulmonary fibrosis(7). Bleomycin administration into rodents leads to the accumula-tion of leukocytes, especially macrophages, followed by the acti-vation of fibroblasts and fibroblast-like cells and the deposition ofcollagen (8–10).

Despite impressive advances in the field, much remains to beunderstood about the source of the fibroblast-like cells that arethought to be responsible for lung fibrosis. One hypothesis is thatlocal interstitial lung fibroblasts migrate into the affected area and

produce extracellular matrix proteins, leading to fibrosis (1). Analternative hypothesis is that in addition to migration of local fi-broblasts, circulating bone marrow-derived fibroblast precursorspresent within the blood are attracted to sites of injury, where theydifferentiate into spindle-shaped fibroblast-like cells called fibro-cytes and at least in part mediate tissue repair (11, 12). Fibrocytesexpress markers of both hemopoietic cells (CD45, MHC class II,CD34) and stromal cells (collagen-I, collagen-III, and fibronectin)(11, 13, 14). Fibrocyte precursors appear to differentiate from asubpopulation of CD14� peripheral blood monocytes (14–16).

In irradiated mice engrafted with GFP-expressing bone marrowcells, bleomycin installation into the trachea causes fibrosis in thelungs, and the fibrotic lesions contained GFP-expressing cells (17).Very few GFP-expressing cells were found in the lungs of controlmice. The GFP-expressing cells in the lungs of the bleomycin-treated mice expressed collagen-I and the chemokine receptorsCCR7 and CXCR4, which are also expressed by fibrocytes (14, 15,17). When cultured in vitro, the GFP-expressing cells also had thespindle-shaped morphology of fibrocytes (17). Installation of FITCinto the lung also causes pulmonary fibrosis in mice, and this fi-brosis also involves the recruitment of bone marrow-derived fi-brocytes (18). Injections of mature fibrocytes into bleomycin- orFITC-treated mice augment pulmonary fibrosis (12, 19). Theseobservations indicate that bone marrow-derived fibrocytes partic-ipate in pulmonary fibrosis.

We previously found that fibrocyte differentiation is inhibited bythe plasma protein serum amyloid P (SAP)3 (3, 15, 20, 21). SAPis a member of the pentraxin family of proteins that include C-re-active protein (CRP). SAP is not related to serum amyloid A, se-rum amyloid protein, or amyloid precursor protein (15, 20, 22).SAP is a 27-kDa protein that is produced by the liver, secreted into

*Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005;and †Department of Experimental Radiation Oncology, University of Texas M.D.Anderson Cancer Center, Houston, TX 77030

Received for publication October 24, 2006. Accepted for publication June 29, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Howard Hughes Medical Institute, Grant 005/04from the Scleroderma Foundation, Grant C-1555 from the Robert A. Welch Foun-dation, and National Institutes of Health Grants CA64193 and HL083029.2 Address correspondence and reprint requests to Dr. Darrell Pilling, Department ofBiochemistry and Cell Biology, MS-140, Rice University, Houston, TX 77005-1894.E-mail address: dpilling@rice.edu

3 Abbreviations used in this paper: SAP, serum amyloid P; �-SMA, �-smooth muscleactin; CRP, C-reactive protein; DAPI, 4�,6�-diamidino-2-phenylindole; pulse Ox, per-centage of hemoglobin saturated with oxygen.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

The Journal of Immunology

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the blood, and circulates as stable 135-kDa pentamers (23, 24).SAP binds to apoptotic cells, DNA, and certain microorganisms,and is cleared by macrophage-like cells through Fc�Rs (25–28). Inhumans and rats, the levels of SAP in the serum are maintained atrelatively constant levels, with CRP acting as an acute-phase re-sponse protein (23). In mice, SAP acts as an acute-phase protein,and CRP is at low steady-state levels (23, 29). However, human,rat, and mouse SAP bind to the same molecules, although withdiffering affinities, indicating a functional similarity (30, 31). In-jections of SAP into humans, mice, and rats appear to have notoxic effects (32, 33).

The ability of SAP to inhibit fibrocyte differentiation in vitro,and the observation that fibrocytes play a role in animal models oflung fibrosis, suggested that SAP might be able to reduce fibrocyteaccumulation or fibrocyte differentiation in vivo, and thus reducelung fibrosis. We report in this work that SAP injections in rats andmice diminish bleomycin-induced lung fibrosis.

Materials and MethodsProduction of murine and rat SAP

Native rat or murine SAP was prepared from commercially available serum(Sigma-Aldrich or Gemini Bio-Products) using calcium-dependent bindingto phosphoethanolamine-conjugated agarose, following standard purifica-tion techniques (33–35). Rat SAP was then repurified using pneumococcalC-polysaccharide-coated beads to remove any excess CRP (35). The pu-rified rat or mouse SAP was diluted with 20 mM sodium phosphate buffer(pH 7.4) and concentrated with a 15-ml centrifugal filter device(UFV2BGC40; Millipore). This procedure was repeated to obtain 500�g/ml SAP in 20 mM sodium phosphate buffer (pH 7.4). Endotoxin levelswere tested using the Limulus amebocyte lysate assay (E-Toxate; Sigma-Aldrich) and were also tested using HEK293 cells transfected with the LPSreceptors CD14 and TLR4 (HEK-Blue LPS detection kit; InvivoGen). En-dotoxin levels were always below detectable levels. Analysis of the SAP byCoomassie- and silver-stained polyacrylamide gels (Bio-Rad) revealedonly one band at 25–27 kDa under reducing conditions, and a single 130-to 135-kDa band under nonreducing conditions (Fig. 1 and data notshown). The identity of the protein (either rat or mouse SAP) was con-firmed by Western blotting using goat anti-rat SAP or sheep anti-mouseSAP polyclonal Abs (R&D Systems), as described previously (15) (Fig. 1).BenchMark protein and MagicMark XP m.w. markers (Invitrogen LifeTechnologies) were used for reducing and Western blot gels, respectively.BSA (Pierce), commercially purified human CRP, human SAP, and murineSAP (EMD Biosciences-Calbiochem), and rat rCRP (R&D Systems) wereused as loading controls.

Animal models of pulmonary fibrosis

Lung fibrosis was induced in 150-g male Sprague-Dawley rats (CharlesRiver Laboratories) with an intratracheal instillation of 100 �l of 10 U/mlbleomycin (EMD-Calbiochem), as previously described (36). Lung fibrosiswas induced in 20-g male C57BL/6 mice (The Jackson Laboratory) with anintratracheal instillation of 60 �l of bleomycin (3 U/kg). All animals wereused in accordance with rules published by the National Institutes ofHealth. The Rice University Institutional Animal Use and Care Committeeapproved the rat procedures (which were performed at Rice University),and the M.D. Anderson Cancer Center Institutional Animal Use and CareCommittee approved the mouse procedures (which were performed atM.D. Anderson Cancer Center).

Injections of SAP

Following bleomycin instillation, rats were injected with an i.p. injection of240 �g of rat SAP or an equal volume of sodium phosphate buffer the dayafter bleomycin treatment and again on days 3, 5, 7, and 9. Alterna-tively, rats were injected with SAP on days 7, 9, 11, and 13. Mice weretreated daily, starting the day after bleomycin injection, with an i.p.injection of either 50 �g of murine SAP or an equal volume of sodiumphosphate buffer. Intraperitoneal injections of SAP reach the bloodwithin 1 h (30, 37).

Measurement of peripheral blood oxygen content

To assess peripheral blood oxygen content in vivo, rats were monitored forthe percentage of hemoglobin saturated with oxygen (pulse Ox). Rats werebriefly sedated with 4% isoflurane in 4 L/min oxygen. Rats were then

removed to room air, and a peripheral pulse Ox sensor (9847V monitorwith a 2000SL sensor; Nonin Medical) was attached to the left rear paw.Pulse Ox readings were taken as the animal regained consciousness.

Quantification of collagen

Rats and mice were euthanized at day 14 or 21 after bleomycin instillation,and the lungs were perfused by injections of PBS into the right ventricle ofthe heart to remove blood. For rats, the whole right lung was removed,weighed, and minced into small pieces, and whole lung collagen contentwas assessed by the Sircol collagen assay (Biocolor), according to themanufacturer’s instructions (12). For mice, collagen was assessed on for-malin-fixed paraffin-embedded sections of whole lung, as described previ-ously (38, 39). Briefly, 15-�m sagittal (longitudinal from top to bottom)sections were cut, and 10 sections from across the lung were used to quan-tify collagen content. Sections were deparaffinized, and then incubated for30 min at room temperature with a saturated solution of picric acid indistilled water containing 0.1% fast green FCF and 0.1% Sirius red F3BA(Polysciences). Sections were repeatedly rinsed with distilled water, andthe dye was eluted with a mixture of 0.1 N NaOH and absolute methanol(1:1, v/v). Spectrophotometer readings were taken at 540 and 605 nm (cor-responding to the maximum absorbance of Sirius red and fast green, re-spectively). The absorbances were used to calculate the amount of collagenand noncollagenous protein in the samples. Collagen content is expressedas a percentage of total protein.

Immunohistochemistry

For rats, the left lung was inflated in prewarmed OCT (VWR) and thenembedded in OCT, frozen on dry ice, and then stored at –80°C. Immuno-histochemistry was performed, as described previously (40, 41). Briefly,10-�m cryosections were mounted on Superfrost Plus microscope slides(VWR). Sections were fixed in acetone for 10 min at room temperature.Nonspecific binding was then blocked by incubation in 4% BSA (fractionV, globulin free; Sigma-Aldrich) in PBS for 60 min. Slides were thenincubated with 5 �g/ml primary Abs in PBS containing 4% BSA for 60

FIGURE 1. Preparation of rat and murine SAP. A, Purified rat SAP wasanalyzed by PAGE, on a 4–15% reducing gel, and stained with Coomassie.M, BenchMark m.w. markers. Lanes 1–5, 1000, 300, 100, 30, and 10�g/ml BSA-loading controls. Lanes 6–10, 1000, 300, 100, 30, and 10�g/ml human SAP-loading controls. Lanes 11 and 12, 1/1000 and 1/300dilutions of purified rat SAP. B, A Western blot of purified rat SAP wasstained with goat anti-rat SAP Abs. M, MagicMark m.w. markers. Lanes1–3, 10, 1, and 0.1 �g/ml human SAP. Lanes 4–6, 100, 10, and 1 �g/mlrat CRP. Lanes 7–9, 1000-, 300-, and 100-fold dilutions of purified ratSAP. C, Purified mouse SAP was analyzed by PAGE on a 4–15% reducinggel, and stained with Coomassie. M, BenchMark m.w. markers. Lanes 1–3,5-, 20-, and 10-fold dilutions of purified mouse SAP. Lanes 4–8, 150, 100,75, 50, and 25 �g/ml BSA-loading controls. D, A Western blot of purifiedmouse SAP was stained with sheep anti-mouse SAP Abs. M, MagicMarkm.w. markers. Lanes 1–3, 1% human, mouse, and rat serum. Lanes 4 and5, 1 �g of commercial human SAP and CRP, respectively. Lanes 6–8, 1,0.3, and 0.1 �g/ml commercial mouse SAP-loading controls. Lanes 9–11,1, 0.3, and 0.1 �g/ml commercial rat CRP-loading controls. Lane 12, 1�g/ml purified mouse SAP.

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min. Rat lung sections were stained for CD32 (D34-485, mouse IgG1; BDBiosciences) to detect B cells, neutrophils, and monocytes/macrophages;CD45 (OX-1, mouse IgG1; BD Biosciences) to detect all leukocytes; CD68(ED1, mouse IgG1; Serotec) as a pan-macrophage marker; and �-smoothmuscle actin (�-SMA) (clone 1A4, mouse IgG2a; Sigma-Aldrich) wasused to detect activated fibroblasts and fibrocytes. Collagen-I was stainedwith rabbit polyclonal Abs (600-401-104; Rockland). Isotype-matched ir-relevant mouse mAbs or irrelevant polyclonal Abs (BD Biosciences, R&DSystems, or Jackson ImmunoResearch Laboratories) were used as controls.

Slides were then washed in six changes of PBS over 30 min and incu-bated for 30 min with 2.5 �g/ml biotinylated rat F(ab�)2 anti-mouse IgG(Jackson ImmunoResearch Laboratories) or biotinylated goat F(ab�)2 anti-rabbit IgG (Southern Biotechnology Associates) in PBS containing 4%BSA, as appropriate. After washing, the biotinylated Abs were detectedwith a 1/200 dilution of ExtrAvidin alkaline phosphatase (Sigma-Aldrich)in PBS containing 4% BSA. Staining was developed with the Vector RedAlkaline Phosphatase Kit (Vector Laboratories) for 10 min. Sections werethen counterstained for 10 s with Gill’s hematoxylin No. 3 diluted 1/5 withwater (Sigma-Aldrich), and were then rinsed in water. Slides were dehy-drated through 70, 95, and 100% ethanol; cleared with xylene; andmounted with Permount (VWR).

For immunofluorescence staining, following incubation with primaryAbs, as described above, or �-SMA directly conjugated to Cy3 (clone 1A4;Sigma-Aldrich), slides were labeled with biotinylated rat F(ab�)2 anti-mouse IgG or Rhodamine Red-X donkey F(ab�)2 anti-rabbit IgG (JacksonImmunoResearch Laboratories). After washing, the biotinylated Abs weredetected with streptavidin-Alexa 488 (Invitrogen Life Technologies-Mo-lecular Probes) in PBS containing 4% BSA. Sections were then fixed for 30min in 70% ethanol containing 0.1% Sudan Black B to quench tissueautofluorescence (42). Slides were then washed in PBS and mounted withVectaShield containing 4�,6�-diamidino-2-phenylindole (DAPI; VectorLaboratories). All procedures were at room temperature.

The number of positive cells was assessed by point counting 10 selectedfields per section with a random start position, as described previously (40,43–47). Sections were analyzed from the top, middle, and lower portionsof each lung, generating 30 regions of analysis per lung. Results wereobtained from at least two individuals blinded to the identity of the sec-tions. Analysis of sections stained with Abs to �-SMA or collagen wasanalyzed with ImageJ software using standard algorithms to define theareas of staining, after first excluding areas containing vessels that contain

�-SMA or collagen (Rasband, W. S., ImageJ, U.S. National Institutes ofHealth, Bethesda, MD).

Fibrosis scoring in mice

Following euthanasia of mice, the lungs were perfused with 10% neutralbuffered formalin, fixed in formalin overnight, and then embedded in par-affin. For each mouse, the lung lobes were separated before being embed-ded together in paraffin to facilitate analysis. Sectioning, staining withH&E, and video imaging to identify fibrosis in the lung were performed, aspreviously described (48–51). The fibrosis score from this technique cor-relates with the score from noninvasive microcomputed tomography scans(52). Results were obtained from two individuals blinded to the identity ofthe sections.

Statistics

Statistical analysis was performed using GraphPad Prism software. Differ-ences between two groups were assessed by Student’s t test. Differencesbetween multiple groups were assessed by ANOVA using Tukey’s post-test. Significance was defined as p � 0.05. In the figures, � indicates p �0.05; �� indicates p � 0.01; and ��� indicates p � 0.001.

ResultsSAP injections reduce bleomycin-induced pulmonary fibrosisin rats

To test the ability of SAP to reduce fibrosis, rats were given anintratracheal instillation of bleomycin (day 0) to induce pulmonaryfibrosis. To reduce the possible effects of SAP interacting withbleomycin, we used a single injection of bleomycin rather than a2-wk continuous dose via osmotic pumps (48). Bleomycin is rap-idly cleared from the body of mammals with a t1/2 of �60 min, soonly residual levels of bleomycin were likely to be present in theanimals when the SAP injections were started on day 1 (53–56).Rats were then treated with either five injections of 240 �g of ratSAP or phosphate buffer every 2 days (days 1, 3, 5, 7, and 9).

FIGURE 2. SAP injections reduce bleomycin-induced histological changes in rat lungs. A, Cryosections from day 14 rat lungs were stained with H&Eto show cellular inflammation, or picrosirius red to show total collagen deposition. Sections were also stained with anti-collagen-I or anti-�-SMA Abs. Barsare 200 �m. Bottom row, Sections were also dual labeled with anti-�-SMA Abs (green) and the nuclear counterstain DAPI (blue), at higher magnificationto show �-SMA expression in individual cells. Bar in bottom row is 50 �m. Photomicrographs are representative sections of three to eight animals pergroup. Saline, intratracheal instillation of saline and then injections of phosphate buffer; Bleo, intratracheal instillation of bleomycin and then injections ofbuffer; Bleo � SAP, intratracheal instillation of bleomycin and then SAP injections; Saline � SAP, intratracheal instillation of saline and then SAPinjections. B–D, Low magnification images of cryosections from day 14 rat lungs. Sections were stained with picrosirius red to show collagen deposition.B, Intratracheal instillation of saline; C, intratracheal instillation of bleomycin, and arrows point to areas of fibrosis. D, Intratracheal instillation of bleomycinand then SAP injections. Bar is 2 mm.

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Other rats were given an intratracheal instillation of saline,and then received either SAP or buffer injections. As previouslyobserved, bleomycin instillation caused a disruption of the nor-mal lung architecture, especially collapsed alveoli, along withareas of inflammatory cell infiltrates and foci of fibroblasts, asdetermined by H&E staining of cryosections (Fig. 2). There wasmuch less disruption of the lung architecture when rats wereinjected with SAP following bleomycin instillation. Comparedwith the saline instillation and buffer injection, lungs from ratsinjected with SAP following saline instillation were indistin-

guishable as determined by double-blind observations (Fig. 2and data not shown).

A key marker of bleomycin-induced lung fibrosis is excessivecollagen deposition. As expected, as compared with the saline in-stillation control, bleomycin instillation led to an elevation in col-lagen content in the lungs as assessed by picrosirius red staining,whole lung collagen levels, and immunohistochemical staining forcollagen-I (Figs. 2 and 3, A and B). Collagen levels in the lungs ofrats injected with SAP following bleomycin instillation were sig-nificantly lower than collagen levels in the lungs of rats injected

FIGURE 3. SAP injections reducebleomycin-induced changes in ratlungs. A, Whole right lungs were re-moved at day 14, and assessed for col-lagen content. Values are means �SEM (n � 4–5 per group). Comparedwith saline, bleomycin instillation led toa significant elevation in collagen levelsas determined by Student’s t test. Com-pared with bleomycin instillation, intra-tracheal instillation of bleomycin andthen SAP injections had a significant re-duction in collagen levels as determinedby Student’s t test. B and C, Sectionswere stained with anti-collagen-I (B) oranti-�-SMA Abs (C), and the percent-age area stained was quantified as a per-centage of the total area of lung. Valuesare means � SEM (n � 3–6 rats pergroup). Significance was determined byANOVA. D, Rats (n � 6 per group)were assessed for peripheral blood ox-ygen content (pulse Ox) at day 14, andcompared with values from 25 healthyage-matched male rats (baseline). Val-ues are means � SEM. Significancewas determined by ANOVA.

FIGURE 4. SAP injections reduce bleo-mycin-induced leukocyte infiltration in ratlungs. Cryosections of rat lung were labeledwith Abs against CD32 (Fc�RII-bearing Bcells, neutrophils, and macrophages), CD45(total leukocytes), and CD68 (macrophages).A, Representative photomicrographs of lungsat day 14. Bars are 200 �m. B–D, Quantifi-cation of the numbers of cells stained forCD32 (B), CD45 (C), and CD68 (D). Valuesare means � SEM (n � 3–6 rats per group).Significance was determined by ANOVA.

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with saline following bleomycin instillation (Figs. 2 and 3, A andB). Injections of SAP in saline-instilled rats had no obvious effecton collagen levels in the lungs.

Another marker of lung fibrosis is the increased expression of�-SMA, a marker for activated fibroblasts and fibrocytes (14, 20,57). As expected, intratracheal instillation of bleomycin led to anincrease in the expression of �-SMA compared with the salineinstillation control (Figs. 2 and 3C). There was an increase in the

number of individual �-SMA-positive cells in the alveoli of ratsfollowing instillation of bleomycin (Fig. 2). Levels of �-SMA inthe lungs of rats injected with SAP following bleomycin instilla-tion were significantly lower than the levels in bleomycin/saline ratlungs (Figs. 2 and 3C). Injections of SAP in saline-instilled ratshad no obvious effect on �-SMA in the lungs.

Pulse oximetry is a simple, noninvasive method to determineperipheral blood oxygen content, and is widely used in the

FIGURE 5. SAP injections reducethe number of bleomycin-induced fi-brocytes in rat lungs. A, Representa-tive photomicrographs of lungs at day14. Cryosections of rat lungs werelabeled with Abs to collagen-I (red)and CD45 (green), and were coun-terstained with DAPI (blue). Bar is200 �m. B, Regions from A athigher magnification show individ-ual cells dual labeled (yellow) forcollagen-I and CD45. Bar is 50 �m.C and D, Quantification of the num-bers of cells stained for collagen-Iand CD45 (C), or �-SMA and CD45(D). Values are means � SEM, n �3– 6 per group. Significance was de-termined by ANOVA.

FIGURE 6. SAP injections reduce bleo-mycin-induced pulmonary fibrosis in mice.A, Percentage change in body weight inmice given intratracheal bleomycin alone(Bleo) or intratracheal bleomycin plusSAP (Bleo � SAP), compared with age-matched saline controls (Saline). B and C,SAP injections decrease pulmonary fibro-sis (B) and percentage collagen content(C) in lungs of bleomycin-treated mice.Values are means � SEM, n � 6. Signif-icance was determined by ANOVA. D, Pho-tomicrographs of H&E-stained sections ofwhole lungs from individual mice instilledwith saline, bleomycin, or bleomycin, andthen injected with murine SAP. Arrowspoint to areas of fibrosis. Each lung lobe wasseparated before being embedded in paraffinto facilitate analysis. Bars are 2 mm.

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management of critically ill patients. To assess peripheral bloodoxygen content in vivo, rats were monitored for the percentage ofhemoglobin saturated with oxygen (pulse Ox). Intratracheal instil-lation of bleomycin led to a reduction in pulse Ox readings ascompared with saline controls (Fig. 3D). Pulse Ox readings of ratsinjected with SAP following bleomycin instillation were similar toboth the levels in saline control rats and baseline measurementsfrom 25 healthy age-matched rats. Together, our data indicate thatSAP injections reduce the severity of bleomycin-induced pulmo-nary fibrosis.

SAP injections reduce bleomycin-induced leukocyte infiltrationinto rat lungs

Besides collagen deposition, bleomycin instillation induces a pro-found leukocyte infiltration into lungs (9, 12, 17, 58). Therefore,we assessed whether SAP injections led to an alteration of leuko-cyte numbers in rat lungs 14 days after bleomycin instillation.Bleomycin instillation induced significant increases in the numberof CD32-, CD45-, and CD68-positive leukocytes, compared withsaline controls (Fig. 4). The increased numbers of CD32-, CD45-,and CD68-positive cells were present not only in areas of col-lapsed alveoli and fibrosis, but also in the interstitial space betweenalveoli. Compared with bleomycin/saline, there were decreasednumbers of CD32-, CD45-, or CD68-positive cells in lungs fromrats injected with SAP following bleomycin instillation (Fig. 4).Injections of SAP in saline-instilled rats had no obvious effect onleukocyte infiltration in the lungs. These data indicate that SAPinjections reduce bleomycin-induced CD32-, CD45-, and CD68-positive leukocyte infiltration in rat lungs.

SAP injections reduce the number of bleomycin-inducedfibrocytes in rat lungs

We also assessed whether fibrocyte numbers were altered follow-ing SAP injections. CD45 and collagen-I dual-positive cells (fi-brocytes) were elevated following bleomycin instillation (Fig. 5,A–C), as described previously (12, 19). Compared with bleomycin/saline, there was a significant decrease in the number of CD45/collagen-I dual-positive cells in lungs from rats injected with SAPfollowing bleomycin instillation (Fig. 5, A–C). Injections of SAPin saline-instilled rats had no obvious effect on the number ofCD45/collagen-I-positive fibrocytes.

Double labeling with �-SMA and CD45 to identify activatedfibrocytes indicated that bleomycin instillation led to a significantincrease in the number of activated fibrocytes compared with sa-line controls (Fig. 5D). As above, compared with bleomycin/sa-line, there was a significant decrease in the number of activatedfibrocytes in lungs from rats injected with SAP following bleomy-cin instillation (Fig. 5D). Compared with saline controls, injectionsof SAP in saline-instilled rats had no statistically significant effecton activated fibrocytes in the lungs. Together our data indicate thatSAP injections reduce the bleomycin-induced increase in the num-ber of fibrocytes and activated fibrocytes in rat lungs.

SAP injections reduce bleomycin-induced pulmonary fibrosisin mice

To test whether SAP injections were effective in another species,we repeated these studies in mice. Compared with bleomycin-treated mice not given SAP, mice injected with murine SAP afterbleomycin instillation had significantly less weight loss (Fig. 6A).The pulmonary fibrosis induced by bleomycin instillation in miceinjected with SAP was significantly lower compared with bleomy-cin-treated mice not injected with SAP (Fig. 6B). Collagen levelsin the lungs of mice injected with murine SAP following bleomy-cin instillation were similar to the levels in control rat lungs (Fig.

6C). Analysis of whole lung sections indicates that there was noapparent fibrosis in the saline-treated mice (Fig. 6D, top panels).The mice instilled with bleomycin had profound fibrosis, both ad-jacent to the bronchi, and at distal sites (Fig. 6D, middle panels).The mice instilled with bleomycin and then injected with SAP hada much reduced level of fibrosis, which was usually restricted toareas near the bronchi (Fig. 6D, lower panels). Together, thesedata indicate that SAP injections decrease bleomycin-induced lungfibrosis in mice as well as rats.

Delayed SAP injections reduce bleomycin-induced pulmonaryfibrosis in rats

To test the efficacy of SAP treatment on an ongoing fibrotic re-sponse, rats were given an intratracheal instillation of bleomycin(day 0) to induce pulmonary fibrosis, as described above. We then

FIGURE 7. Delayed SAP injections reverse bleomycin-induced pulseOx levels. Rats (n � 6–7 per group) were assessed for peripheral bloodoxygen content (pulse Ox) at days 7 (A), 14 (B), and 21 (C). Values aremeans � SEM. Significance was determined by ANOVA.

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delayed treatment with SAP until day 7, which would likely reducethe possible effects of SAP interacting with apoptotic material gen-erated by bleomycin instillation during the first 12–48 h (59–61).

Rats were treated with injections of 240 �g of rat SAP on days 7,9, 11, and 13. Intratracheal instillation of bleomycin led to a re-duction in pulse Ox readings at days 7, 14, and 21, as compared

FIGURE 8. Delayed SAP injections reduce bleomy-cin-induced fibrosis in rat lungs. A, Whole right lungswere removed at day 21 and assessed for lung collagencontent. Values are means � SEM (n � 6–7 per group).B–F, Cryosections from day 21 rat lungs were stainedwith picrosirius red to show collagen deposition. B, In-tratracheal instillation of saline; C and D, intratrachealinstillation of bleomycin; E and F, intratracheal instil-lation of bleomycin and then delayed SAP injections.Asterisks indicate areas of fibrosis. Bars are 2 mm.

FIGURE 9. Delayed SAP injections reduce bleomycin-in-duced inflammation in rat lungs. A–C, Cryosections from day21 rat lungs were stained with CD45 to show cellular inflam-mation. A, Intratracheal instillation of saline; B, intratrachealinstillation of bleomycin; C, intratracheal instillation of bleo-mycin and then delayed SAP injections. Bars are 200 �m.D–F, Cryosections of rat lung were labeled with Abs againstCD32, CD45, and CD68. Quantification of the numbers ofcells stained for CD32 (D), CD45 (E), and CD68 (F). Valuesare means � SEM (n � 4 for controls, n � 6 for bleo andbleo � SAP).

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with saline controls (Fig. 7). Pulse Ox readings of rats that wereto be injected with SAP on days 7, 9, 11, and 13 followingbleomycin instillation had reduced pulse Ox readings at day 7(before SAP treatment began), indicating that these animals re-ceived a similar insult to the bleomycin-untreated rats (Fig. 7A).However, at days 14 and 21, the pulse Ox readings of ratstreated with delayed SAP injections were significantly higherthan the bleomycin/saline readings (Fig. 7, B and C). These dataindicate that delayed SAP injections reduce bleomycin-inducedlung dysfunction.

As seen with rats at day 14, rats at day 21 had an elevation incollagen content in the lungs as assessed by whole lung collagenlevels (Fig. 8A) and picrosirius red staining (Fig. 8, B–F). Collagenlevels in the lungs of rats injected with SAP from days 7 to 13following bleomycin instillation were similar to the levels in con-trol rat lungs (Fig. 8A). Analysis of low power images of lungsections indicated that there was no apparent fibrosis in the saline-treated rats (Fig. 8B). The rats instilled with bleomycin had exten-sive fibrosis (Fig. 8, C and D). The rats instilled with bleomycinand then injected with SAP had a much reduced level of fibrosis(Fig. 8, E and F).

We also assessed whether delayed SAP injections affect leuko-cyte infiltration in rats following bleomycin instillation. At day 21,bleomycin instillation induced significant increases in the numberof CD32-, CD45-, and CD68-positive leukocytes, compared withsaline controls (Fig. 9). Compared with bleomycin/saline, delayedinjections of SAP caused a significant decrease in the numbers ofCD32-, CD45-, and CD68-positive cells in the lungs (Fig. 9, D–F).These data indicate that the delayed SAP injection protocol wasalso able to reduce bleomycin-induced CD32-, CD45-, and CD68-positive leukocyte infiltration in rat lungs.

DiscussionWe have shown that injections of a plasma protein, SAP, are ableto decrease bleomycin-induced lung fibrosis in both rats and mice.In rats, SAP injections significantly reduced the increase in num-bers of leukocytes, fibrocytes, and activated fibrocytes associatedwith bleomycin-induced lung fibrosis. SAP injections not only re-duced pathological changes in lung morphology, but also main-tained peripheral blood oxygen content, as assessed by pulse Oxmeasurements.

The concentrations of SAP injected into rats and mice weredesigned to only double the normal physiological dose of SAP.Rats have �30 �g/ml circulating SAP levels, and C56BL/6 micehave �10–20 �g/ml SAP (29, 35, 62). Assuming that a 150-g rathas �8 ml of serum and the serum concentration of SAP in a ratis � 30 �g/ml, 240 �g of SAP will approximately double theserum level of SAP (35). Similarly, a 20-g mouse has �1.4 ml ofblood, and the serum concentration of SAP in C57BL/6 mice is�15 �g/ml, so 50 �g of murine SAP will approximately doublethe serum level of SAP (62).

We previously found that SAP inhibits fibrocyte differentia-tion (15, 20, 21). Fibrosis involves fibrocytes, so the ability ofSAP injections to reduce bleomycin-induced pulmonary fibrosismay be due to SAP inhibiting fibrocyte differentiation. Alter-natively, SAP may reduce fibrosis by reducing the accumulationof mature fibrocytes or their precursors. However, we have alsoobserved that SAP reduces the bleomycin-induced increase inthe number of leukocytes in lungs. Because fibrocytes secretecytokines that promote leukocyte recruitment (63), and SAPdecreases the accumulation of fibrocytes, SAP may reduce lev-els of these inflammatory cytokines, and thus reduce leukocyterecruitment. A second possibility is that SAP may directly in-

hibit the recruitment of leukocytes into lungs following bleo-mycin instillation. A third possibility is that because SAP bindsapoptotic material, treatment with SAP may accelerate clear-ance of apoptotic material following instillation of bleomycinor during fibrosis (61). As the presence of apoptotic materialrecruits leukocytes, the rapid removal of this material by SAPmay decrease leukocyte recruitment after bleomycin instillationor during fibrosis (64).

The reduced fibrosis in rats treated with the delayed SAP treat-ment protocol indicates that SAP may be able to affect an ongoingfibrosis. The treatment of SAP from days 7 to 13 would most likelypreclude the effect of SAP binding to either bleomycin or apoptoticmaterial generated during the acute stage of bleomycin toxicity(53–56, 59–61). The peak of leukocyte and fibrocyte accumula-tion in lungs following bleomycin or FITC instillation occurs be-tween days 7 and 14 (12, 13, 18, 65). Our data indicate that treat-ment with SAP during this time either inhibits the differentiation offibrocyte precursors into fibrocytes in situ, or prevents their initialaccumulation in the lung.

Besides pulmonary fibrosis, there is now clear evidence thatfibrocytes are involved in other fibrosing diseases. In humans, fi-brocytes have been detected in tumors, skin wounds followingeither burns or incisions, hypertrophic scars, bronchial asthma, andnephrogenic fibrosing dermopathy (11, 65–67). In animal models,fibrocytes are associated with experimental fibrosis induced by ir-radiation damage, bleomycin injections into the skin, intimal hy-perplasia of the carotid artery, systemic acetaminophen (paraceta-mol) administration, unilateral ureteral obstruction, bile ductligation, chronic granuloma formation following Schistosoma ja-ponicum infection, and skin wounding (11, 68 –72). We havealso recently found that fibrocytes are involved in cardiac fi-brosis following ischemia-reperfusion injury in mice, and thatSAP inhibits this fibrosis (21). These studies clearly indicatethat bone marrow-derived fibrocytes are involved in manyforms of fibrosis. We have shown in this study that SAP injec-tions reduce bleomycin-induced pulmonary fibrosis in both ratsand mice. These results suggest that SAP may be useful as anantifibrotic therapy for pulmonary fibrosis.

AcknowledgmentsWe thank Varsha Vakil, Kathleen MacKay, and Kelly Campbell for ex-cellent technical assistance, and Deen Bakthavatsalam for critical readingof the manuscript.

DisclosuresRice University has patent applications on the use of SAP to inhibit fibro-sis, and this intellectual property has been licensed to Promedior.D. Pilling and R.H. Gomer are founding members of, have equity in, andreceive royalties from Promedior.

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