monocytes may amplify their recruitment into inflammatory lesions
TRANSCRIPT
78
Monocytes May Amplify Their RecruitmentInto Inflammatory Lesions by Inducing
Monocyte Chemotactic Protein
Susan D. Cushing and Alan M. Fogelman
By Northern analysis, freshly isolated monocytes contained no detectable mRNA for monocytechemotactic protein-1 (MCP-1). However, after 4 hours of incubation at 37°C, MCP-1 mRNAwas clearly induced in the monocytes and was found to be highly dependent and directlyproportional to the monocyte density. The level of MCP-1 mRNA continued to increase,reaching a peak after 22 hours of incubation. After 3 days in culture, MCP-1 mRNA levels haddeclined substantially and after 8 days were undetectable in the monocytes/macrophages. Theamount of MCP-1 protein secreted correlated with the density-dependent increase in MCP-1message. We hypothesize that the migration of monocytes into inflammatory lesions may beamplified by the density and time-dependent induction of MCP-1. (Arteriosclerosis andThrombosis 1992;12:78-82)
The infiltration of circulating blood monocytesacross vascular endothelium is an importantearly event in the inflammatory response
that is common to a variety of injurious conditions,including infection, cancer, atherogenesis, and trau-ma.1-8 This monocyte infiltration and the subsequentevents that lead to the acute and chronic presence ofinflammatory macrophages within infected or dis-eased tissue are probably regulated by a series ofrapid environmental changes.9 These changes mostlikely begin with a local elaboration of a chemoat-tractant to mediate accumulation of monocytes at thesite of inflammation. Previous studies have demon-strated that endothelial cells10'11 and smooth musclecells12-13 in culture constitutively express monocytechemotactic protein-1 (MCP-1).1415 Recently, wehave shown that MCP-1 accounts for virtually all ofthe monocyte chemotactic activity produced by en-dothelial cells and smooth muscle cells, alone and incoculture.16 The results of this study suggest thatafter the initial recruitment by cells of the vessel wall,monocytes may be involved in the amplification oftheir own recruitment into inflammatory lesions byinducing MCP-1.
From the Division of Cardiology, Department of Medicine,UCLA School of Medicine, Los Angeles, Calif.
Supported by National Institutes of Health grants HL-30568 andRR 865, Bethesda, Md., and by the Laubisch, Rachel Israel Berro,and Milt Grey Funds. S.D.C. is a Nassi Fellow.
Edwin L. Bierman kindly acted as Guest Editor for this article.Address for correspondence: Dr. Susan D. Cushing, Depart-
ment of Medicine, UCLA School of Medicine, Room 47-123 CHS,Los Angeles, CA 90024-1679.
Received January 29, 1991; revision accepted October 23, 1991.
MethodsCell Culture
Human monocytes and lymphocytes were ob-tained by a modification of the Recalde method.17
The monocytes were suspended in Iscove's modifiedDulbecco's medium containing 30% autologousserum, 1% glutamine, 1% penicillin/streptomycin/fungizone, and 0.0022% insulin (supplemented me-dia) and then plated at varying densities in plasticculture dishes or treated lipopolysaccharide-freeTeflon pots. The cells were incubated at 37°C from4 hours to 8 days. The media were endotoxin free18'19
and were changed every 3-4 days.
Northern Blot AnalysisTotal RNA was isolated from the monocytes/mac-
rophages by the guanidinium isothiocyanate methodof Chomczynski and Sacchi.20 The alcohol-precipi-tated RNA was electrophoresed on formaldehyde/1%agarose gels and transblotted to Biotrans nylon mem-branes (Bio-Rad, Richmond, Calif.). The blots werehybridized with a 32P-end-labeled 35-mer oligonucle-otide probe. The probe was complementary to nucle-otides 257-291 of the published cDNA sequence forMCP-1.21 The sequence of the probe was 5-CGGAT-G T T G G G T T T G C T T G T C C A G G T G G T C -CATGG-3'. Hybridization was done overnight at 60°Cin a solution containing 0.75 M NaCl, 0.15 M tris(hy-droxymethyl)aminomethane hydrochloride (pH 8.0),10 mM EDTA, 5xDenhardt's solution (lxDen-hardt's solution: 0.02% bovine serum albumin/0.02%Ficoll/0.02% polyvinylpyrrolidone), 0.1% sodium do-decyl sulfate, 0.1% Na2P2O7, 50 fig heat-denatured,
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Cushing and Fogelman Induction of MCP-1 79
a- tubulinPLASTIC TEFLON
MCP-1
TIME Oh 4h Id 5d 8d
CELL DENSITY
xlO4/em2 6 12 45
a-tubulin
MCP-1
5 15 SO
Oh 4h I d 5d Sd
TIME IN CULTURE
FIGURE 1. Expression of monocyte chemotactic protein-](MCP-1) mRNA during monocyte in vitro differentiation.Monocytes were plated at a density of20xlO'< cells/cm2 for thetime shown on the x axis. Panel A: RNA was extracted, andNorthern blot analysis was performed with MCP-1 and a-tu-bulin probes. Panel B: Laser densitometry scans of Northernblots of MCP-1 mRNA normalized for a-tubulin. h, Hours; d,days; A U, absorbance units.
sheared salmon sperm DNA per milliliter, and 2x10*cpm of probe per milliliter. Blots were washed twicewith 20 ml 0.2xstandard saline citrate (lxstandardsaline citrate: 0.15 M NaCl/15 mM sodium citrate) and0.1% sodium dodecyl sulfate at 60°C for 30 seconds,washed twice with 500 ml of the same solution at 60°Cfor 30 minutes, dried, and exposed for 16-64 hours(within the linear range of the film) to Kodak XAR-5film with an intensifying screen at -70°C. The autora-diographs were scanned on an LKB Ultrascan laserdensitometer (LKB, Piscataway, N.J.) and quantifiedwith the GELSCAN n program for the Apple II computer.
Determination of Monocyte ChemotacticProtein-1 Protein
The media from 1-day-old monocyte culturesplated at varying densities were analyzed for MCP-1protein using MCP-1 purified from baboon smooth
m
> 20
12 45 5 15 50
CELL DENSITY x 10 ' /cm 2 CELL DENSITY x 1 0 ' t o n l
FIGURE 2. Density-dependent expression of monocytechemotactic protein-1 (MCP-1) mRNA. Monocytes wereplated either in plastic culture dishes (to which they avidlyadhere) or in tipopolysaccharide-frce Teflon pots (to whichthey do not readily adhere) at the densities shown on the x axis.Panel A: RNA was extracted, and Northern blot analysis wasperformed with MCP-1 and a-tubulin probes. Panel B: Laserdensitometry scans of Northern blots of MCP-1 mRNA nor-malized for a-tubulin. AU, absorbance units.
muscle cells as a standard and an antibody toMCP-1, both provided by Anthony J. Valente.22
The media were centrifuged through Centricon 30concentrators to remove the high-molecular-weightserum proteins. Then the eluant was concentratedusing Centricon 3 concentrators. The final mediumvolume was 10 /xl per million cells plated. Theamount of MCP-1 protein in a 50- ,̂1 aliquot ofconcentrated media was quantified by slot-blotanalysis using Amersham's Enhanced Chemilumi-nescence (ECL) immunodetection system, Hybond-ECL nitrocellulose, and Hyperfilm (Amersham, Ar-lington Heights, 111.). The recommended ELCblotting protocol was followed. The primary anti-body against MCP-1 was diluted 1:1,000, and thesecondary antibody (anti-rabbit immunoglobulinG-POD, from Boehringer Mannheim, Indianapolis,Ind.) was diluted 1:5,000. The films generated fromthe slot-blot analysis were scanned on an LKB
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80 Arteriosclerosis and Thrombosis Vol 12, No 1 January 1992
5 10 20 40
CELL DENSITY xlO4/cm2
FIGURE 3. Bar graph of density-dependent expression ofmonocyte chemotactic protein-1 (MCP-1) protein. Monocyteswere plated in plastic culture dishes and incubated at 37 °Cfor1 day at the densities shown on the x axis. The media werecollected, concentrated, assayed for MCP-1 protein by slot-blotanalysis, and quantified by densitometry as described in"Methods."
Ultrascan laser densitometer and quantified withthe GELSCAN II program for the Apple II computer.
Results and DiscussionMononuclear cells mediate inflammatory re-
sponses, in part by the release of biologically activemolecules whose production is regulated by the stageof cellular maturation.23-24 Therefore, we examinedperipheral blood monocytes for the expression ofMCP-1 mRNA throughout in vitro differentiation.As shown in Figure 1, freshly isolated monocytescontained no detectable mRNA for MCP-1. How-ever, after 4 hours in culture at 37°C, MCP-1 messagewas clearly induced, reaching a maximal level at 24hours; thereafter it steadily declined, falling to pre-culture levels by 8 days. Because the infiltration ofmonocytes into a region of inflammation is an earlyevent in the inflammatory process, the observed timecourse of induction of MCP-1 mRNA is consistentwith the idea that monocytes play a role in their ownrecruitment into an area of inflammation. Because13-25% of the cells in the monocyte preparationwere lymphocytes, isolated lymphocyte preparationswere cultured and tested for MCP-1 mRNA. Inpreparations from five different donors, no MCP-1message was seen (data not shown). Also, MCP-1mRNA expression by 4-hour- and 1-day-old culturesof monocytes plated at a constant density with theproportion of lymphocytes ranging between 13% and66% was examined. The amount of MCP-1 messageproduced was again dependent on time in culture butwas independent of the proportion of lymphocytes inthe culture (data not shown).
Because it has been shown that the expression ofcolony stimulating factor-1, tumor necrosis fac-
CELL DENSITY
x lO 4 /em 2 0 5 10 20 40
E
3
CO
>cc
ioCO
zHIQ
10 20 40
CELL DENSITY x 10 \ c m 2
FIGURE 4. Effect of media conditioned by monocytes platedat various densities on the expression of monocyte chemotacticprotein-1 (MCP-1) mRNA by monocytes plated at a uniformdensity. Monocytes were plated at the densities shown on the xaxis and incubated for 4 hours at 37°C, and then the media(conditioned media) were removed and placed on othermonocytes that were freshly plated at the uniform density of20xl04 cells/cm2. Panel A: RNA was extracted from cellsexposed to conditioned media, and Northern blot analysis wasperformed with MCP-1 and a-tubulin probes. Panel B: Laserdensitometry scans of Northern blots of MCP-1 mRNA nor-malized for a-tubulin. AU, absorbance units.
tor-a, lysozyme, c-frns, and platelet-derived growthfactor(B) mRNA in cultured monocytes is adher-ence regulated,2526 the effect of adherence on theexpression of MCP-1 mRNA was determined byculturing monocytes in Teflon pots, to which theydo not readily adhere,27 as well as in plastic culturedishes, to which they avidly adhere. The effect ofcell density on the expression of MCP-1 messagewas also tested. Figure 2 demonstrates that theexpression of MCP-1 mRNA in cultured monocytesis density dependent but not dependent on celladhesion. Monocytes plated at similar densities ineither plastic dishes or Teflon pots expressed equiv-alent amounts of MCP-1 mRNA. Although therewas some donor variability in expression of MCP-1mRNA, density regulation of message expression
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Cushing and Fogelman Induction of MCP-1 81
was observed in five of five donors. The maximalexpression of message was seen at a cell density of40-50xlO4/cm2. Figure 3 shows that the secretionof MCP-1 protein correlated with the density-de-pendent expression of MCP-1 message.
To determine if the density regulation of MCP-1 inmonocytes was due to secretion of some factor thatinduced the message, monocytes were plated atvarious densities and incubated at 37°C for 4 hours;the media (conditioned media) were removed andplaced on monocytes that were freshly plated at a lowand uniform density. If monocytes had secreted afactor that was responsible for the density regulationof MCP-1, the conditioned media taken from thecells plated at varying densities should have con-tained corresponding amounts of the factor andshould have produced corresponding inductions ofMCP-1 message. However, Figure 4 demonstratesthat this was not the case, as monocytes that wereexposed to unconditioned media and conditionedmedia taken from cells plated at varying densities (upto 40xl04/cm2) contained the same amount ofMCP-1 mRNA. These results suggest that excretionof a soluble factor is not involved in the induction ofMCP-1 mRNA in monocytes.
Yla-Herttuala and coworkers28 have shown thatMCP-1 mRNA was expressed in early human andrabbit atherosclerotic lesions in areas where thedensity of monocytes/macrophages was high.Neiken et al29 also observed that the majority ofMCP-1 production was in macrophages and thatMCP-1 was noted in all regions of the plaque withhigh concentrations of macrophages. Based onthese observations Neiken et al29 hypothesized that"The production of MCP-1 by macrophages mayrepresent an amplification mechanism for the re-cruitment of additional macrophages to the athero-sclerotic plaque." The results of our study areconsistent with this hypothesis.
AcknowledgmentWe thank Cynthia Harper for excellent technical
assistance.
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S D Cushing and A M Fogelmanchemotactic protein.
Monocytes may amplify their recruitment into inflammatory lesions by inducing monocyte
Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1992 American Heart Association, Inc. All rights reserved.
Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 GreenvilleArteriosclerosis, Thrombosis, and Vascular Biology
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