Proc. Natl. Acad. Sci. USAVol. 90, pp. 6086-6090, July 1993Medical Sciences

Early and persistent induction of monocyte chemoattractantprotein 1 in rat cardiac allografts

(arteriosclerosis/cardiac transplantation/macrophage/early response gene)

MARY E. RUSSELL*tli, DAVID H. ADAMStt, LAURI R. WYNERt, YUKARI YAMASHITA*, NANCY J. HALNON*,AND MoRRis J. KARNOVSKYt*Harvard School of Public Health, tHarvard Medical School, and *Brigham and Women's Hospital, Boston, MA 02115

Communicated by Eugene Braunwald, April 12, 1993 (received for review December 23, 1992)

ABSTRACT The early response gene for monocytechemoattractant protein 1 (MCP-1) encodes a potent chemo-tactic factor that is specific for monocytes. To determinewhether MCP-1 is involved in macrophage recruitment incardiac allografts, we studied time-dependent MCP-1 gene andprotein expression patterns in the heterotopic, Lewis to F-344rat transplantation model (by reverse transcription-PCR andimmunohistochemistry). There was a significant increase (8- to12-fold) in MCP-1 gene transcripts in cardiac allografts com-pared with host hearts at 7, 14, and 28 days after transplan-tation. This induction was not observed with syngeneic trans-plants or hosts exposed to the same circulating cells and bloodproducts. The MCP-1 gene product was expressed predomi-nantly by mononuclear cells that double-stained with anti-macrophage antibody (ED1) and localized to the interstitial andvascular spaces of the allografts. Immunocytochemical cellcounting revealed significant increases in both MCP-1- andEDl-immunopositive cells in 7-, 14-, and 28-day allografts (incomparison with day 0 hearts). The absolute number ofMCP-1-positive cells (5-7%) was lower than that of EDl-positive cells (25-34%) at all time points, suggesting thatMCP-1-positive cells represent a subpopulation of activatedmacrophages. The persistent expression of MCP-1 in associa-tion with increased macrophage localization suggests that thisinducible mediator contributes to the chronic inflammatoryresponse following cardiac transplantation and that it may playa role in the pathogenesis of transplant arteriosclerosis.

Chronic rejection manifested by transplant arteriosclerosis isthe leading cause of late graft failure after cardiac transplan-tation (1). The persistent infiltration of monocytes-macrophages into the interstitium and vessels of chronicallyrejecting cardiac allografts (2-4) suggests that these cells,once activated, produce a host of active cytokines andgrowth factors that may mediate the inflammatory response(5-7).Monocyte chemoattractant protein 1 (MCP-1) is a potent

chemotactic factor specific for monocytes and is encoded byan early response gene (8). In vitro studies have demon-strated that expression of MCP-1 is induced by interferon y,tumor necrosis factor, and platelet-derived growth factor inmonocytes, endothelial cells, and smooth muscle cells, re-spectively (8, 9). Furthermore, MCP-1 transcriptional andtranslational products have been identified in vivo within theatherosclerotic regions of blood vessels from humans, rab-bits, and hypercholesterolemic primates (10-12) as well aswithin the proliferative intimal lesions associated with bal-loon injury (9).To investigate the potential role of MCP-1 in monocyte-

macrophage recruitment after cardiac transplantation, we

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

studied MCP-1 expression patterns in a heterotopic abdom-inal heart transplantation model (4). We used the Lewis toF-344 rat model because it results in long-term graft survivaland arteriosclerotic thickening in allograft vessels similar tothat seen in humans. The vascular changes include adhesionofmonocytes to the vessel lumen at 15 days, accumulation ofmonocytes-macrophages in the neointima at 45-75 days, andintimal expansion due predominantly to smooth muscle cellsat 120 days (3). We used reverse transcription-PCR to ana-lyze cardiac tissues for MCP-1 gene expression and immu-nohistochemistry to study MCP-1 protein expression at var-ious points after transplantation. Our characterization ofMCP-1 in this in vivo model documents the linkage ofmacrophage activation and the pathogenesis of chronic re-jection and arteriosclerosis.

MATERIALS AND METHODSRat Heart Transplantation. Heterotopic abdominal cardiac

allografts (Lewis to F-344 rat) were transplanted as described(4). The allograft study group included 9 host (F-344) hearts(3 for each point after transplantation; 7, 14, and 28 days) and12 allografted (Lewis) hearts (3 for each time point and 3day-0 hearts that were harvested but not transplanted). Wealso performed syngeneic control transplantations (Lewis toLewis) to assess the contribution of surgical manipulation tothe inflammatory response. Thus, all grafted hearts wereLewis so that the differences observed were not due togenetic variations in donor strain. In this syngeneic versusallogeneic study group, there were four transplanted rats (twoallogeneic and two syngeneic). These animals generated twoLewis syngrafts, two Lewis syngeneic host hearts, two Lewisallografts, and two F-344 allogeneic host hearts, all eight ofwhich were harvested 14 days after transplantation. At thetime of harvesting a 4-mm section was removed from themidventricle of each heart for histologic analysis, and theremaining ventricle was used for RNA extraction.

Reverse Transcription-PCR. Total cellular RNA was ex-tracted from ventricular sections with RNazol B (CINNA/Biotecx Laboratories) according to the manufacturer's in-structions. First-strand cDNA synthesis using 2.5 mg of totalRNA was completed with Moloney murine leukemia virusreverse transcriptase (GIBCO/BRL), according to the man-ufacturer's instructions, and then diluted 1:4.

Oligonucleotides were synthesized by Genosys Biotech-nologies (The Woodlands, TX). The primer sequences for ratMCP-1 were chosen to span exon/intron borders (ATG CAGGTC TCTGTC ACG for the 5' end and AGTATGACAGAGAAC TAG for the 3' end) (13). Acidic ribosomal phospho-protein PO (ribo) was used as an internal reference to reflect

Abbreviations: MCP-1, monocyte chemoattractant protein 1; ribo,ribosomal phosphoprotein P0.§To whom reprint requests should be addressed at: Harvard Schoolof Public Health, 677 Huntington Avenue, Boston, MA 02115.

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total cellular RNA because it represents a ubiquitouslyexpressed mRNA whose protein levels are under transla-tional control (14). The primer sequences for ribo were TCATTG TGG GAG CAG ACA for the 5' end and AAA GGAAGA GTC GGA GGA for the 3' end, resulting in an 834-bpproduct.PCR studies were carried out on a GeneAmp system 9600

(total vol, 25 ,l); the "hot start" technique (15) was used toincrease specificity. Reaction conditions included 5 ,ul ofcDNA, 1 ,uM (each) 5' and 3' primers, 10 mM Tris HCl/50mM KCI/1.5 mM MgCl2/0.001% (wt/vol) gelatin/800 ,uMdNTPs/0.625 unit of AmpliTaq DNA polymerase. The ther-mal cycling parameters were denaturation at 94°C for 15 sec,annealing at 50°C for 20 sec, and extension for 60 sec (witha final extension of 7 min at the end of all cycles). PCRproducts (10 j4) were analyzed on 3% agarose gels. Weperformed negative control experiments for each primer pairby substituting water for cDNA or omitting reverse tran-scriptase during cDNA synthesis. After optimizing primerselection and PCR conditions for each gene of interest, wedirectly cloned the amplified products into the TA vectorPCRII (Invitrogen, San Diego). Sequence analysis (Seque-nase v2.0 kits; United States Biochemical) by the dideoxy-nucleotide chain-termination method confirmed amplifica-tion of the expected MCP-1 and ribo fragments, with >90%ohomology to the sequences submitted to the GenBank database. Identity was also confirmed by Southern blot analysisof amplified PCR products, by using 32p-, 5'-end-labeledoligonucleotides internal to the original PCR primers.

Quantification of PCR Products. When it was necessary toquantitate PCR-amplified fragments the reaction was carriedout with 300,000 cpm of [32P]dCTP (800 Ci/mMol; 1 Ci = 37GBq; DuPont/NEN) per tube. Agarose gels (3%) containingthe amplified products were dried and exposed to storagephosphor plates for 8-16 hr. Incorporated 32p was measuredon a PhosphorImager system (Molecular Dynamics, Sunny-vale, CA) (16). The system's volume integration feature wasused to express total radioactivity in each amplified PCRproduct band in PhosphorImager units. Specific radioactivityin each band was calculated by subtracting the backgroundradioactivity for that gel lane. To ensure that the initial targetmRNA represented a portion of the amplified product, wedetermined the linear range of amplification for each primerpair as a function of cycle number and template or cDNAconcentration. For MCP-1, PCR was performed on serialdilutions of cDNA for 28 cycles (for ribo, 26 cycles). Therelative level of mRNA transcripts for MCP-1 and ribo wasthen measured in all hearts in each experimental group withinthese linear PCR ranges.Each study group was subjected to a minimum of three

PCR analyses in duplicate. To determine whether measuredamplified products represented significant differences in theallograft study group, we used two-factor analysis of variance(ANOVA) without replication. We used one-way ANOVAfor statistical analysis in the syngeneic versus allogeneicstudy and for comparisons of trends over time. When theANOVA was significant, individual comparisons were madewith Student's t test and the significance level for the P valueswas corrected by the Bonferroni method. Corrected MCP-1values were derived by dividing 32p measured in the MCP-1band for each sample by that in the ribo band (using the meanvalue from the duplicate measurements) to normalize forpotential differences in total cellular RNA in the PCR anal-yses and to allow relative comparisons between samples inexperimental sets. Corrected MCP-1 data were expressed asthe mean ± SEM for each sample after all analyses had beencompiled.Immunohistochemical Studies. Ventricular specimens were

serially sectioned into 2-mm slices, fixed for 4 hr in methanol/Carnoy's solution, and embedded in paraffin. Sections (4 ,um)

were immunostained for MCP-1 with a primary rabbit poly-clonal antibody (1:500 dilution for 1.5 hr at 25°C) recognizingJE antigen (the murine equivalent of MCP-1), which waskindly provided by Barrett J. Rollins (Dana-Farber CancerInstitute, Boston) (17). The secondary, biotinylated antibodywas goat anti-rabbit. Negative controls included (i) omissionof the primary antibody; (ii) use of an alternative primaryantibody, unrelated to MCP-1/JE, that was directed againstfactor VIII; and (iii) preincubation of the antibody withJE-conditioned medium from COS cells transfected withmouse JE cDNA (1:50 dilution) or with control conditionedmedium obtained from cells transfected with the cDNA in theantisense direction (18). As a positive control, we demon-strated that the murine anti-MCP-1 antibody identifiedMCP-1 protein in rat aortic smooth muscle cells (a-actinpositive) that had been maintained in vitro and serum stim-ulated. Immunostaining for cellular characterization was alsocompleted as described (3) with monoclonal antibodies thatrecognize (i) rat monocyte-macrophages (ED1; Serotec); (ii)rat T cells (W313; Accurate Chemicals, Westbury, NY); and(iii) smooth muscle a-actin (anti-smooth muscle cell a-actin;Sigma). For the double-labeled immunoenzyme experiments,we used avidin-biotin techniques to identify the first anti-body, followed by an alkaline phosphatase reaction (VectorLaboratories) to identify the second antibody (19). To quan-titate the percentage of MCP-1- and anti-macrophage (ED1)-immunopositive cells in cardiac allografts at various timepoints, we scored 500 cells (nuclei) in random high-poweredfields (x40) in the most densely stained areas of each heartsection.

RESULTSReverse Transcription-PCR. The linear range of amplifica-

tion with respect to cycle number was established for eachprimer pair (Fig. 1 A and B). cDNA dilution studies (Fig. 1C,MCP-1; Fig. 1D, ribo) identified the linear portion for each

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Total RI/A(ng)FIG. 1. Linear PCR amplification of MCP-1 and ribo gene tran-

scripts. Rat cardiac allograft cDNA was amplified in the presence ofMCP-1 primers (A and C) and primers for a ubiquitously expressedribosomal control gene (ribo; B and D). Amplified products wereseparated electrophoretically and 32p incorporation was measured inPhosphorImager units. (A and B) Linear relation between PCR cyclenumber and amplified MCP-1 or ribo. (C and D) Linear relationbetween various concentrations of added cDNA (represented as thecalculated amount of total RNA in the PCR) and amplification ofMCP-1 at 28 cycles or ribo at 26 cycles.

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gene, which was used to determine relative differences inmRNA.

Fig. 2 shows a representative PCR analysis comparingmRNA transcript levels for MCP-1 (Fig. 2A) and ribo (Fig.2B) in the allograft study group. Fig. 2 (Top) shows amplifiedPCR products visualized by staining the agarose gels withethidium bromide. For MCP-1, the amplified product bandsare intense in the allografts, whereas they are barely visiblein the host and day-0 hearts. For ribo there is little variationin band intensity among samples. Fig. 2 (Middle) shows thesame dried agarose gels visualized by exposure to storagephosphor plates, and Fig. 2 (Bottom) shows incorporation of32p as measured in Phosphorlmager units. These valuesrepresent composites from the three sets of experimentscompleted in duplicate on each heart. Although there wasvariation in absolute values between experimental sets com-pleted on different days, relative differences between heartswere preserved. For each experimental set, two-wayANOVA identified a significant difference between MCP-1values for amplified product from allografts and host hearts(P < 0.001), and a t test confirmed differences at each timepoint (P < 0.005). For the reference gene ribo, there wasvariation between samples for amplified product bands but nosignificant difference between samples in the host or trans-plant groups. Similar results were obtained with ,B2-microglobulin (data not shown), suggesting that the varia-tions in ribo levels were due to differences in RNA loading.

Corrected MCP-1 Transcript Levels. Fig. 3A shows cor-rected MCP-1 transcript levels in the allograft study group,where each bar represents the mean ± SEM from six PCRanalyses of a single heart. Given the mean (days 7-28)corrected MCP-1 value of 0.7 in the allografts compared withthat of 0.07 in the host hearts (P < 0.0001), this differencerepresents a 10-fold induction. Significant increases inMCP-1 transcript levels in the transplanted compared withhost hearts are also apparent at all time points (7, 14, and 28days) (P < 0.005). Comparisons across time in the allograftgroup revealed a significant difference between the day-0Lewis control point (0.031 ± 0.005) and each of the laterLewis to F-344 experimental points (day 7, 0.885 ± 0.21; day14, 0.623 ± 0.08; day 28, 0.628 ± 0.11) (P < 0.008). The

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differences between days 7, 14, and 28, however, were notstatistically significant, suggesting that, after initial induc-tion, MCP-1 transcript levels do not change with time.

In comparing the syngeneic and allogeneic study groups,we identified a significant increase in corrected MCP-1 tran-script levels for allografts versus syngrafts (Fig. 3B) (P <0.0001) but found none in comparing the two host groups.Differences in corrected MCP-1 transcript levels betweenallografts and allogeneic host hearts were significant (P <0.006), whereas those between syngrafts and either hostgroup were not. The increase in MCP-1 transcript levels inallografts but not syngrafts or host hearts suggests that thisstimulation arises from an immune response related to thegenetic mismatch between Lewis and F-344 rats.

Immunohistochemical Studies in Allografts. Histologic sec-tions from Lewis day-0 hearts showed preservation of normalcardiac structure. Smooth muscle cell staining with a-actinwas limited to the media, and T-cell (W313) and MCP-1staining were typically absent in these control hearts. Sectionsfrom day-7 allografts showed early, patchy interstitial accu-mulations of mononuclear cells, many of them EDl-positivemonocytes or W313-positive T cells. There was early adhesionof EDl-positive cells to the arterial lumen (Fig. 4A), and cellspositive for MCP-1 were usually limited to areas focallyinfiltrated by mononuclear cells. The findings for day-14allografts were similar, with more diffuse interstitial infiltra-tion by mononuclear cells, monocyte and T-cell adhesion tothe vessel lumen, and MCP-1 staining limited to mononuclearcells. The day-28 allografts showed moderate graft infiltrationwith mononuclear cells. Many EDl-positive monocytes, someMCP-1-positive mononuclear cells, and some W313-positiveTcells were present throughout the interstitium. There wasdiffuse intimal thickening in some arteries consisting predom-inantly of EDl-positive monocytes, some of which stainedpositive for MCP-1 (Fig. 4B). T cells and smooth muscle cellswere infrequently observed in the intima. In addition, werarely identified MCP-1 staining in spindle-shaped cells of thesmooth muscle type (in venules) or in the endothelium (invenules). Lewis syngrafts removed after 14 days showedpreservation of cardiac and vascular structure, with the oc-casional localization of interstitial mononuclear cells (usually

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FIG. 2. PCR amplification of MCP-1 and ribo transcripts from allograft and host hearts after cardiac transplantation. PCR amplificationproducts were compared between RNA extracted from host hearts and transplanted (allograft) hearts at 7, 14, and 28 days after transplantation.Donor hearts that were harvested but not transplanted are designated day 0. (Top) Representative ethidium bromide-stained agarose gels showingPCR products. (Middle) Radioactive images of the same gels reflecting 32p incorporated in the amplified product bands. (Bottom) Compositesof six PCR analyses showing incorporated radioactivity (in PhosphorImager units) in amplified product bands. Data are plotted as means + SEM.Each bar represents PCR analyses in a single heart compiled from at least three sets of experiments completed in duplicate. (A) MCP-1 levelsin transplanted hearts at 7, 14, and 28 days increased significantly in comparison with host and day-0 hearts (P < 0.006). (B) Ribo levels variedbetween samples at days 7, 14, and 28, but there was no significant trend between host and transplanted hearts.

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FIG. 3. Corrected MCP-1 levels in the allograft heart group atvarious times after transplantation and in the syngeneic versus

allogeneic group. MCP-1 PCR values were normalized against thosefor the control ribosomal gene and are presented as corrected MCP-1levels in relative units. (A) Allograft study group. There is an increasein corrected MCP-1 levels in allograft compared with host hearts at7, 14, and 28 days after transplantation (P < 0.005) and at day 0compared with days 7, 14, and 28 (P < 0.008). (B) Syngeneic versusallogeneic study group. Corrected MCP-1 levels are significantlyelevated in cardiac allografts compared with syngrafts (P < 0.0001)or with host hearts from syngeneic or allogeneic transplantations (P< 0.001). Data are derived from at least three separate experimentscompleted in duplicate, with at least two animals in each group.

to the subendocardium). Most of these cells were ED1 posi-tive; a few were MCP-1 positive. Double-labeling studiesperformed on cardiac allograft tissue showed that 90% of theMCP-1 protein expression was in EDl-positive monocytes-macrophages (Fig. 4C). Double-stained, MCP-1-expressingmonocytes were located more frequently in the interstitiumand less frequently in the intima.

Fig. 5 shows that the number of MCP-1- and EDl-positivecells increased significantly after transplantation in compar-ison with that in day-0 Lewis hearts. Although the absolutenumber of cells expressing MCP-1 was small, it increasedsignificantly between day 0 (mean ± SEM, 0.6% ± 0.1%) anddays 7 (5% ± 1%), 14 (7% ± 2%), and 28 (7% ± 2%) (n = 3for each point; P < 0.02). Because we counted nuclei thatreflected the entire cell population, it is likely that some cellsdeemed negative were fibroblasts or myocytes. The absolutenumber of EDl-positive monocytes-macrophages washigher than that of MCP-1-positive cells, and it too increasedsignificantly in allografts (day 0, 4% ± 1%; day 7, 34% ± 3%;day 14, 34% ± 1%; day 28, 25% ± 1%) (n = 3 for each point;P < 0.001). The proportion of EDl-positive cells expressingMCP-1 (=25%) did not change at the various time points.

DISCUSSIONWe establish in this in vivo study that MCP-1 is induced at themRNA and protein levels in a series of animals at various

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times after cardiac transplantation and that the induction isspecific to allografted tissue. We also provide data in supportof the previously unconfirmed assumption that immune stim-ulation after cardiac transplantation is chronic and involvesactivated macrophages.Our use ofthe heterotopic Lewis to F-344 rat model has the

advantages of simulating the human condition (with long-term graft survival and the development of chronic rejectionwith arteriosclerotic lesions) and of producing two heartssubject to the same circulatory system: the diseased allograftand the disease-free host heart. We demonstrate by semi-quantitative PCR experiments that induction of MCP-1 genetranscripts in the cardiac allograft is early (within 7 days of

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FIG. 5. EDl- and MCP-1-immunopositive cells at different timesafter cardiac transplantation. Immunocytochemical cell countingcompleted for macrophages (ED1) (hatched bars) and MCP-1 (stip-pled bars) is presented as percentage of positive cells from 500 nucleicounted in random high-powered fields (x40) from the most denselystained areas. Compared with day 0, there are significant increasesin cells staining for both MCP-1 and ED1 at days 7, 14, and 28.However, after the initial induction there was no significant changewith time. *, P < 0.001; 1, P < 0.02.

transplantation) and persistent (extending through 28 days).This stimulation appears to be an allogeneic phenomenon, inlight of our observation that MCP-1 transcript levels were notelevated either in matched F-344 host hearts subject to thesame circulating cells and blood products or in Lewis syn-grafts subject to the same surgical procedure but matched fordonor and recipient.Dallman et al. (20) have identified differing cytokine ex-

pression patterns in allografts, syngrafts, and normal heartsat early points in a murine acute cardiac rejection model. Forexample, interleukins la and 3 were upregulated in bothsyngrafts and allografts, whereas interleukins 2 and 4 andtumor necrosis factor 8 were upregulated only in allografts.In our chronic cardiac rejection model, MCP-1 induction andarteriosclerotic changes both occurred exclusively in al-lografts. The factors responsible for MCP-1 induction in thisin vivo model have yet to be identified. Given the inflamma-tory milieu of the cardiac allograft and findings from previousin vitro studies, possible candidates include interferon y,

platelet-derived growth factor ,3 chain, and interleukins 1 and4 (8).MCP-1 mRNA has been identified by Nelken et al. (10) in

human atheromas, by Yla-Herttuala et al. (11) in hypercho-lesterolemic rabbit aortas 10 weeks after balloon injury, andby Yu et al. (12) in arteries ofhypercholesterolemic monkeys.The induction of MCP-' in our model of transplant arterio-sclerosis extends these earlier studies implicating MCP-1, byvirtue of its identification in diseased vessels, in the patho-genesis of naturally occurring atherosclerosis. Using immu-nohistochemistry, we show that mRNA induction in cardiacallografts correlates with greater numbers of cells stainingpositive for MCP-1. In Nelken's study (10) of atheroscleroticregions from human carotid endarterectomy specimens,MCP-1 mRNA was detected in 5-33% of cells counted. Thesmaller percentage (5%) of MCP-1-positive cells detected inour study may reflect technical differences in staining be-tween in situ RNA hybridization and immunohistochemis.ryfor a secreted gene product, or it may simply relate todifferences in species or disease process.

In concert with observations in human atheromas (10, 12),we found that most MCP-1-positive cells were macrophages.In our study, MCP-1-positive cells represented a subpopula-tion (==25%) of the EDl-positive macrophages. Thus, at leastsome macrophages localizing to the cardiac allograft were"activated" in the sense that they expressed MCP-1, an

inducible gene product. In addition, we demonstrate anincrease in the total number of macrophages in allogeneicallytransplanted cardiac tissue. One possible explanation is thatthere is a positive feedback mechanism in the cardiac al-lograft that causes MCP-1-producing monocytes to "amplifythe recruitment" of additional monocytes (10, 21).The biologic significance ofMCP-1 in cardiac allografts has

not been established. The most obvious role, as suggested byits name, is to regulate the chemotaxis of monocytes to theheart and vessels and thereby amplify the immune responsesthat they engender. MCP-1 is also expressed in diseasedvessels, where the pathologic hallmark is smooth muscle cellaccumulation; this raises the possibility that MCP-1 regulatessmooth muscle cell chemotaxis as well (22). Finally, MCP-1can activate macrophages and alter their function (as mani-fest by an increase in their tumoricidal capacity) (8). Ouridentification of a subset of macrophages expressing MCP-1in cardiac allografts provides a foundation for further studiesof macrophage localization and activation in vivo and theinvolvement of macrophages in arteriosclerosis.

We are grateful to Robert J. Schott and John B. Newell forstatistical advice, Tom McVarish for editorial advice, Barrett J.Rollins for his generous gift of MCP-1 reagents and helpful discus-sions, and Edgar Haber for his ongoing support. This work wassupported by a grant from Bristol-Myers Squibb and by NationalInstitutes of Health Grant HL43318.

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2. Salomon, R. N., Hughes, C. C. W., Schoen, F. J., Payne, D. D.,Pober, J. S. & Peter, L. (1991) Am. J. Pathol. 138, 791-798.

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6. Wong, H. L. & Wahl, S. M. (1991) in Peptide Growth Factors andTheir Receptors, eds. Sporn, M. B. & Roberts, A. B. (Springer,New York), pp. 509-548.

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8. Rollins, B. J. (1991) Cancer Cells 3, 517-524.9. Taubman, M. B., Rollins, B. J., Poon, M., Marmur, J., Green,

R. S., Berk, B. C. & Nadal-Ginard, B. (1992) Circ. Res. 70,314-325.

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resis 11, 355-360.17. Rollins, B., Stier, P., Erst, T. & Wong, G. (1989) Mol. Cell. Biol. 9,

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20. Dailman, M. J., Larsen, C. P. & Morris, P. J. (1991) J. Exp. Med.174, 493-496.

21. Cushing, S. D. & Fogelman, A. M. (1992) Arteriosclerosis Thromb.12, 78-82.

22. Marmur, J. D., Friedrich, V. L., Jr., Rossikhina, M., Rollins, B. J.& Taubman, M. B. (1990) Circulation 82, III-698 (abstr.).

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