Materials and Methods. Hyperlipemic Swine Model

Transcription

1 AmericnJournl ofpthology, Vol. 135, No. 2, August 1989 Copyright Americn Assocition ofpthologists Enhnced Monocyte Progenitor Cell Prolifertion in Bone Mrrow of Hyperlipemic Swine Lynn E. Averill,* Richrd C. Megher,t nd Ross G. Gerrity* From the Crdiovsculr Reserch Center, Clevelnd Reserch Institute* nd the Deprtment oflbortory Hemtology, The Clevelnd Clinic Foundtion,t Clevelnd, Ohio This study demonstrtedfor thefirst time tht bone mrrow is trget of enhnced in vivo monocytopoiesis in hyperlipemi. A significntly greter number of bone mrrow cells (BMC) were recovered per femur in swine fed hyperlipemic (HL) diet compred with swinefed norml (N) diet. In ddition, significntly elevted number ofmonocyticprecursorsproliferted in HL-swine compred with N-swine BMC cultures grown in stndrdized medi in the bsence ofn exogenous source ofcolony stimultingfctor (CSF). HL-swine ser stimulted significnt enhncement in the number of proliferting monocytic precursor cells, regrdless of whether or not the BMC were from HL- or N- swine. In ddition, HL-swine compred with N- swine BMC demonstrted n enhnced intrinsic cpcity to form monocytic colonies in culture, irrespective ofthe source ofswine ser used to stimulte growth. (AmJPthol 1989, 135: ) Dietry-induced therosclerosis in hyperlipemic (HL) swine closely simultes tht of the humn in plque morphology nd distribution. Studies in HL-swine from this lbortory1' showed tht res in the swine ort permeble to the protein binding zo-dye, Evns blue,1 re susceptible to erly therosclerotic lesion formtion, wheres djcent impermeble regions re not.2 Atherogenesis in these lesion-susceptible regions involves preferentil recruitment of lrge numbers of blood monocytes into the intim3 where they form fom cells, t lest some of which migrte bck into the blood.4 In this regrd, the monocyte my initilly ply protective role in the therosclerotic process by scvenging lipid from the rtery, thereby reducing plque progression. Recruitment of monocytes into lesion-susceptible regions is medited by monocyte chemotctic fctor specific to monocytes from HLswine.5 Lesion formtion is chronologiclly ssocited with monocytosis, in prt contributed by the ppernce of two unique subpopultions of monocytes in the circultion of HL-swine. Our hypothesis ddresses the concept tht enhnced recruitment of monocytes into lesion-susceptible rteril regions of HL-swine is fcilitted by incresed prolifertion of monocytic progenitor cells in the bone mrrow, with the subsequent relese of elevted numbers of monocytes into the circultion. In this study, we demonstrted tht the bone mrrow is trget of enhnced monocytopoiesis under hyperlipemic conditions. First, significntly greter number of bone mrrow cells (BMC) were recovered per femur in HL-swine compred with norml (N) swine. Second, n enhnced number of monocytic progenitor cells were quntitted per femur in HL-swine compred with N- swine, s mesured in stndrd in vitro clonogenic ssy in the bsence of ny exogenous source of colony stimulting fctor (CSF); this suggests tht HL-swine BMC exhibit greter intrinsic cpcity to proliferte nd form monocytic colonies thn do N-swine BMC. Also of considerble importnce ws the finding tht HL-swine ser stimulte prolifertion of monocytic progenitor cells in both N- nd HL-swine BMC cultures. Mterils nd Methods Hyperlipemic Swine Model Ten noncstrted mle Yorkshire swine, 8 to 1 weeks of ge t the initition of the experiment, were divided into Supported by grnt number HL38497 from the Ntionl Hert, Lung, nd Blood Institute nd by the Americn Hert Assocition, Northest Ohio Affilite. Dr. Gerrity is n Estblished Investigtor of the Americn Hert Assocition. Accepted for publiction My 9, Address reprint requests to Ross G. Gerrity, PhD, Scientific Director, Crdiovsculr Reserch Center, Clevelnd Reserch Institute, 2351 Est 22nd Street, Clevelnd, OH

2 37 Averill, Megher, nd Gerrity Tble 1. Enhnced Recovery ofbone Mrrow Cells nd Monocyte Progenitor Cells in Hyperlipemic Swine Diet M-colony/ durtion BMC BMC/femur M-colony/15 femur* (months) donor (X1-8)* BMC plted*t (X1-4) 4 N HL N HL N HL N HL N HL HL BMC donors re significntly greter thn N BMC donors t P <.1 s performed by Wilcoxin's rnk sum test. t M-colony, monocyte colony number per 15 BMC plted s hrvested t 1 dys of culture. two groups: N nd HL. N-swine were plced on stndrd hog msh diet, nd HL-swine were fed the sme msh supplemented with 1.5% cholesterol nd 19.5% lrd.2 Ech of five pirs (one N nd one HL) were killed on different experimentl dys, 4 to 9 months fter diet initition. Before killing, swine were sedted with ketmine hydrochloride (2 mg/kg intrmusculrly) nd nesthetized with Nembutl (Abbott Lbortories, North Chicgo, IL) (intrvenously to effect). Forty ml of blood were drwn from the ven cv into nonheprinized syringe nd processed for collection of serum s described below. Five ml of heprinized (15 U/ml) blood were drwn for totl white cell counts nd differentil counts on ech of the five pirs of nimls. Additionlly, differentil counts were routinely crried out on mtched pirs of nimls used for other experiments t, 12, 15, 18, nd 2 weeks on the diet, s well s when ech niml ws killed. Differentil count dt from four pirs t 2 weeks (including the 2- week pir in Tble 1) were included in this study. At necropsy, ech femur ws dissected free of muscle nd swed in hlf. Bone mrrow ws collected by spirtion nd repeted flushing nd scrping into Hnk's Blnced Slt Solution (HBSS) contining 5% Fetl Bovine Serum (FBS) nd 2 U/ml preservtive-free sodium heprin. Nonheprinized blood (4 ml) ws llowed to clot for 2 hours, the clot loosened with sterile wooden stick, nd the blood ws centrifuged t 3 rpm for 1 minutes. The serum ws decnted, sterile filtered (.45,m), nd stored t -2 C for lter testing in BMC cultures fter determintion of serum cholesterol levels. Bone Mrrow Cell Culture for CFU-GM Bone mrrow suspensions were immeditely processed over cell collector nd nylon mesh funnel to obtin single cell suspensions. BMC were wshed nd resuspended in HBSS contining 5% FBS fter depletion of erythrocytes by mmonium chloride (.83%) lysis. A vible BMC count ws determined in hemocytometer by trypn blue dye exclusion. Preliminry studies demonstrted tht modifiction of the culture techniques of Metclf nd Moore7 optimlly supported the prolifertion of swine BMC precursors tht my form colonies of grnulocytes or monocytes or mixtures of grnulocytes nd monocytes, depending on the source nd concentrtion of CSF. These precursor cells re termed colony-formingunit grnulocyte/monocyte (CFU-GM). Accordingly, stndrd medi ws used to perform ll in vitro clonl ssys in.3% Bcto-gr, consisting of Dulbecco's Modified Egle's Medium High Glucose (DMEM-HG, M. A. Bioproducts, Wlkersville, MD), supplemented with 1% FBS (M. A. Bioproducts), L-glutmine (2 mm), penicillin (1 U/ml), streptomycin (1 8g/ml), 2-mercptoethnol (5 X 1-5 M), DEAE dextrn (4 jig/ml), nd L-sprgine (2 ALg/ml). To test the effects of serum on CFU-GM growth, serum from either HL- or N-swine ws dded to the stndrd medi t concentrtion of 2.5% in combintion with 7.5% FBS. The BMC (15/ml) were plted in 1 ml of gr medium in 35 X 1 mm tissue culture dishes, nd llowed to gel for 1- to 2-minutes before incubting the cultures in 5% C2 tmosphere t 37 C for, 1, nd 14 dys before hrvest. To obtin colony counts, gr cultures were fixed in the petri dish with citrte-cetone-formldehyde, plced on glss slide, dried overnight, nd stined with the combined nonspecific (lph npthyl cette) esterse nd specific (npthol AS-D chlorocette) esterse stining protocols of Sigm kit #91 (Sigm Chemicl Co., St. Louis, MO) for monocytic nd grnulocytic colonies, respectively. Totl numbers of colonies, s well s numbers of ech colony type, were counted for ech gr culture under 1 OCX mgnifiction in n Olympus (Olympus Microscopes, New York, NY) light microscope. Results White Blood Cell Counts As cn be seen in Tble 2, HL-swine (serum cholesterol 4 to 7 mg/dl) fed the cholesterol/lrd-contining diet for 2 weeks demonstrted mrked monocytosis compred with N-swine (serum cholesterol 7 to 1 mg/dl). Reltive percentges of both lymphocytes nd neutrophils were ccordingly decresed. HL-swine tended to hve slightly reduced totl white blood cell (WBC) counts compred with mtched controls, but this difference seldom reched sttisticl significnce. Differences in WBC

3 Bone Mrrow Response to Hyperlipemi 371 Tble 2. Differentil Counts of Whole Blood From Norml nd Hyperlipemic Swine* Lymphocytes Monocytes Neutrophils Bsophils Eosinophils WBC/ml X 1- (%) (%) (%) (%) (%) H-Swine 14.8 ± ± ± 1.t 17.3 ± ±.7.2 ±.1 N-Swine 19.8 ± ± ± ± 1.5. ±.1.2 ±.1 * Hyperlipemic (HL) swine (serum cholesterol 4 to 7 mg/dl) fed cholesterol/lrd-contining diet for 2 weeks demonstrted mrked monocytosis compred with norml (N) swine (serum cholesterol 7 to 1 mg/dl). N = 4 for both HL- nd N-swine. t Significntly greter thn N-swine (P <.1); WBC, totl white blood cells. nd differentils were not seen before 12 to 15 weeks of hyperlipemi, but were observed fter this time in ll nimls with serum cholesterol levels greter thn 4 mg/ dl. No pprent correltion ws observed between the differentil count nd durtion of diet or cholesterol level. BMC Recovery nd Monocyte Progenitor Cell Number The totl number of BMC recovered per femur ws significntly elevted for HL-swine compred with gemtched N-swine (Tble 1). In ddition, the number of monocytic progenitor cells proliferting in 1-dy BMC cultures supported in stndrd medium, with no other exogenous CSF source, ws significntly enhnced for HLswine compred with N-swine (Tble 1). These two phenomen tken together men significntly greter totl number of monocytic progenitor cells in HL-swine compred to N-swine bone mrrow (Tble 1). In ddition, s my be noted in Tble 1, the longer donor pir ws mintined on the diet before killing, the greter the rtio in recovery of HL- to N-swine BMC, lthough n ge-relted decline in the totl numbers of both N- nd HL-swine BMC ws observed. Esterse Stining Ptterns of CFU-GM A difference ws seen in the esterse stining pttern of CFU-GM formed in N- nd HL-swine BMC cultures in response to HL-swine compred with N-swine ser. Three types of colonies were distinguished by the combined nonspecific nd specific esterse stining technique in BMC cultures stimulted with HL-swine ser, nmely, grnulocytic stining, mixed stining, nd monocytic stining. In contrst, the monocytic colony type ws the predominnt type formed in BMC cultures in response to N-swine ser. Individul cells within grnulocytic or monocytic colony types stined uniformly nd chrcteristiclly with either the specific or nonspecific esterse stin, respectively (Figures 1 A nd 1 B). Mixed stining colonies were composed of individul cells tht demonstrted both nonspecific nd specific esterse ctivity. The specific esterse stin ws seen s focl point t one end of the cell, wheres the nonspecific esterse stin ws distributed wekly throughout the cell (Figure 1C). This colony type, therefore, could not be chrcterized s typicl mixed colony composed of both monocytes nd grnulocytes, ie, cells stining exclusively with either the nonspecific or specific esterse stins. On further morphologic exmintion with My-Grunwld nd Giems stins, these mixed stining colonies ppered to be composed predominntly of immture monocytic cells. Numbers nd Types of Colonies Formed Figures 2 nd 3 show the numbers nd types of colonies formed in N- nd HL-swine BMC cultures, respectively, in response to N- or HL-swine ser t, 1, nd 14 dys in culture. An interesting progression of colony types occurred over time in N-swine BMC cultures in response to HL-swine ser (Figure 2). At dys in culture, grnulocytic nd mixed stining colonies were the predominnt colony types, wheres the number of monocytic colonies ws negligible. By 1 dys in culture, however, grnulocytic colonies disppered, mixed stining colonies reched plteu levels, nd monocytic colonies hd incresed mrkedly. Monocytic colonies reched pek levels nd were the only colony type present t 14 dys. Thus, s numbers of grnulocytic nd mixed stining colonies decresed over time in culture, the monocytic colony number incresed. The number nd types of colonies formed in N-swine BMC cultures in response to N-swine ser over time in culture re lso shown in Figure 2. In contrst to the response to HL-swine ser, numbers of grnulocytic or mixed stining colonies were low t ll time points, nd monocytic colonies, the predominnt colony formed in response to N-swine ser, reched plteu by 1 dys t levels significntly lower thn those seen in response to the HL-ser. A similr pftern of colony progression over time in culture ws observed in HL-swine BMC cultures in re-

4 *~~~~~~~~~~~~~~~~~~~~~~~~~....'.;...l....^ Averill, Megher, nd Gerrity 4, * I.. I.., * * *1 ' Figure 1. LigBht microgrph of1-dy swine BMC culture stined with combined specific nd nonspecific esterse stin to differentitegrnulocytic, monocytic, nd mixed stining colonies. The BMC were obtinedfrom HL-swine tht hd been on the dietfor months. : Red rection product ofspecific esterse distinguishes grnulocytic colonies. b: Blck rection product of nonspecific esterse is specific to monocytic colonies. c: Foci of the red rection product of specific esterse, long with diffuse stining ofthe blck rection product or nonspecific esterse in individul cells chrcterizes this mixed stining or erly monocyte colony type (ll figures X 12).

5 Figure 2. Progression of colony types with time in N-swine BMC cultures in response to HL- nd N-swine ser. The dt re representtive ofthe results ofone ofthree experiments performed on BMC recovered from ech ofthree differentn-swine donors tht hd been on the N diet for months. BMC (15/culture) from one N-swine donor wereplted in thepresence ofdifferent smples ofn-swine (N = 4) or HL-swine (N = 4) ser. Duplicte BMC cultures corresponding to ech serum smple were hrvested t, 1, nd 14 dys fter the initition of culture. Numbers of ech colony type were quntitted nd expressed s men ± SEM. : G-colony, grnulocytic colony. b: Mixed stining colony. c: M-colony, monocytic colony. d: Totl CFU-GM mde up of G-, Mixed stining, nd M-colony counts. *P <. 1 s nlyzed by Student 'st-test. u m U Cl c z An OV Bone Mrrow Response to Hyperlipemi 373 () G-Colony (b) Mixed Stlning-Colony - HL-Sor *-* N-Ser T * m (c) M-Colony p p J 1 T / v w s s14,x T* *I \fi (d) Totl CFU-GM *,+* /* _4-_ i~~~~~~~ 14 Dys in Culture * sponse to N- or HL-swine ser (Figure 3). In this cse, however, grnulocytic colony formtion ws negligible in response to either HL- or N-swine ser s mesured t ny time point. Mixed stining colonies predominted t dys in response to HL-swine ser, nd declined to zero between nd 1 dys, with concurrent increse in the number of monocytic colonies. Although the pttern of colony progression ws similr for BMC from N-swine (Figure 2) nd HL-swine (Figure 3), the numbers of mixed stining nd monocytic colonies, s well s the totl CFU- GM formed in the presence of HL-swine ser s indicted t certin time points, were significntly greter thn those found in the presence of N-swine ser. Further experiments confirmed the observtion tht HL-swine ser elicited n enhncement in the number of proliferting CFU-GM in BMC cultures. Figures 4A nd B show the cpbility of N- nd HL-swine BMC, respectively, to form CFU-GM in response to N- or HL-swine ser, s mesured t 1 dys in culture. As Figure 4A shows, the number of mixed stining nd monocytic colonies, s well s totl CFU-GM formed in N-swine BMC cultures, ws significntly greter when stimulted by HL-swine ser compred with N-swine ser. A similr finding ws observed in HL-swine BMC cultures (Figure 4B). We nlyzed our dt further to determine whether there ws n intrinsic difference in the cpbility of HLswine compred with N-swine BMC to proliferte nd form monocytic colonies when stimulted in the presence of either N- or HL-swine ser. As Figure 5A shows, HLswine compred with N-swine BMC demonstrted n enhnced cpbility to proliferte nd form monocytic colonies in response to N-swine ser. Likewise, HL-swine Figure 3. Progression of colony types over time in HL-swine BMC cultures in response to HL- nd N-swine ser. Experimentl design is otherwise like the one identified in Figure 2 for N-swine BMC. *P <. 1 s nlyzed by Student'st -test. ) m ur. ) 4-4DA 8^ () G-Colony _._ f.%]ria^ * 1 14 (b) Mixed Stining-Colony 18 - HL-Ser 1 T*-* N-Ser \. ~~~~~~~ 1 14 x (d) Totl CFU-GM f 12 1 z 8 4 2, t 14 Dys in Culture

6 374 Averill, Megher, nd Gerrity 5 in. W- In 2 UIJ z 1o so go 4 2 =MDM SAMr COLONY m u-coc M IALCPU -O N - SERA N - SERA (A) BMC FROM N-SWINE *HL - SERA 1 DU&% DMU coal PKUM Ul ML-;b-INLI -O_WLI 1 HL- U- SERA I Figure 4. Enhnced stimultory cpbility ofhl-swine compred withn-swine ser to 1 induce monocytic colonyformtion in eiso thern- orhl-swine BMC cultures. The dt re representtive of the results of one of five experiments performed on ech offive different donor pirs ofswine. In ech ex- 4 periment, BMC removedfrom one N-swine nd one HL-swine donor wereplted (1O5/ 2 culture) in the presence of multiple smples of N-swine (N = 4) or HL-swine (N = 4) swine ser. Duplicte BMC cultures corresponding to ech serum smple were 1 hrvested on dy 1. Men ± SEM ws tbulted for ech colony type. A: Colony I so counts for N-swine BMC cultures stimulted in the presence of N- or HL-swine o ser. B: Colony counts for HL-swine BMC cultures stimulted in thepresence ofn- or 4 HL-swine ser. Dt from ech of the five different experiments were nlyzed to- 2 gether by n nlysis ofvrince (ANOVA) resulting in P <. 1for differences in o colonyformtion due to the source ofser (HL versus Nser). BMC demonstrted greter cpcity thn N-swine BMC to form monocytic colonies in response to HL-swine ser (Figure 5B). Discussion In this study, we showed for the first time tht the bone mrrow is trget of hyperlipemi-induced ltered function in vivo, resulting in enhnced monocytopoiesis nd subsequent monocytosis in the swine model of therosclerosis. The number of BMC recovered per femur from HL-swine ws significntly elevted compred with tht from N-swine, indicting tht there re greter numbers of BMC in vivo in swine fed high ft/cholesterol diet. Furthermore, HL-swine BMC produced elevted numbers of monocytic progenitor cells compred with N-swine BMC in vitro under stndrdized culture conditions in the presence of FBS lone. Other thn low levels of CSF, which my be present in FBS, no exogenous CSF ws dded to these BMC cultures. These results suggest tht prior ctivtion of monocyte progenitor cell prolifertion by fctors present in the hyperlipemic environment occurred in vivo. The resulting incresed proportion of proliferting monocytic progenitor cells in HL-swine led to the observed increse in totl number of BMC long with the significnt elevtion in bsolute number of monocytic progenitor cells in vivo. The question of whether the incresed monocyte differentil count reflected increse in the bsolute number of circulting monocytes in ddition to the observed elevted differentil count is currently under study. We designed further experiments to determine whether hyperlipemic serum could stimulte enhnced ~5 ~U to W- N - SMC A) SERA FROM N-SWINE 2 r so 4 = IfDJNI OCLON - U-COLON TOTALOPU -GM m-i I -4- I T l i SERA FROM HL-SWNt'E. * 1so I S4 Figure 5. HL-swine compred withn-swine BMC demonstrtes n enhnced cpbility toform monocytic colonies in response to either N- or HL-swine ser (see legend for Figure 4). A: Colony countsfor N- nd HL-swine BMC in response to N-swine ser. B: Colony countsfor N- nd HL-swine BMC in response to HL-swine ser. The results from five different experiments were nlyzed by ANOVA for differences in the intrinsic cpbility ofhl- nd N-swine BMC to form monocytic colonies in response to either N- or HL-swine ser. The ANOVA resulted in P <.1 for intrinsic differences due to the source ofbmc. z 3 I 1s u -17- N - BMC L.l 3 13

7 Bone Mrrow Response to Hyperlipemi 375 prolifertion of monocytic progenitor cells in N- nd HLswine BMC cultures. A difference ws seen in the esterse stining pttern of CFU-GM formed in BMC cultures in response to HL-swine compred with N-swine ser. Three types of colonies formed in N- nd HL-swine BMC cultures in response to HL-swine ser: grnulocytic, mixed stining, nd monocytic. The mixed stining colony type ws unique in tht it ws not composed of mixture of grnulocytic nd monocytic cells, but of individul cells stining with both nonspecific nd specific esterse stins. These colonies were chrcterized s immture monocytic bsed on morphologic exmintion with My- Grunwld nd Giems stins. In contrst, the monocytic colony type predominted in BMC cultures in response to N-swine ser. An interesting progression of colony types occurred over time in both N-swine nd HL-swine BMC cultures in response to HL-swine ser but not to N-swine ser. HL-swine ser elicited predominntly grnulocytic nd mixed stining colony formtion with little or no monocytic colony formtion t dys in culture. By 1 dys in culture, however, grnulocytic colonies disppered, mixed stining colonies reched plteu levels, nd monocytic colonies incresed remrkbly. Monocytic colonies reched pek levels nd were the only colony type present t 14 dys. This pttern of colony progression ws similr to tht found in BMC cultures derived from other species in tht, given the pproprite source nd concentrtion of CSF, progressive enlrgement in numbers of monocyte colonies over time in culture occurs by trnsformtion of primitive grnulocyte cells into monocyte precursors nd by prolifertion of the monocytic cells. The expnsion of the monocyte popultions is prlleled by reduction in the number of grnulocytic cells either by trnsformtion to monocyte precursors or differentition to short-lived polymorphs.8 In contrst to colony formtion in response to HL-swine ser, only monocytic colonies were formed in pprecible numbers in BMC cultures stimulted in the presence of N-swine ser. Pek numbers were reched between 1 nd 14 dys of culture. Bsed on number nd progression of colony types over time in culture, it would pper tht the mixed stining colonies formed in response to HL-swine ser represent monocytic cells t reltively erly stge of differentition, t lest with respect to the esterse stining pttern. Such colonies differentite over time in culture to the more mture monocytic colony exhibiting only the nonspecific esterse stin. This conclusion is bsed on the observtion tht, s the mixed stining colonies disppered over time in culture, incresing numbers of monocytic colonies ppered nd totl colony number incresed. These mixed stining colonies ppered to hve high prolifertive potentil becuse they were qulittively lrger in dimeter, s noted by visul observtion, reltive to monocytic colonies formed in response to N- swine ser. Indeed, these colonies my be nlogous to the high prolifertive potentil colony forming cell (HPP- CFC) described in other systems.9 HPP-CFC hs been chrcterized s more primitive mcrophge progenitor cell thn the CFU-GM, with the cpbility of giving rise to reltively lrge colonies. Thus, HL-swine ser my contin fctors cpble of inducing the differentition of greter distribution of monocyte precursor cells, including more primitive precursors thn cn be stimulted by N-swine ser. It is of considerble importnce tht HL-swine ser invribly induced greter number of proliferting mixed stining nd monocytic colonies compred with N-swine ser in both N-swine nd HL-swine BMC cultures. This finding suggests tht incresed levels of monocytic (M) or grnulocytic/monocytic (GM) CSF my be present in HL-swine ser. CSF in other systems is glycoprotein required for the prolifertion of monocyte precursor cells.1 In the present cse, CSF my be produced or secreted by cells in vivo in response to fctor in HL-swine ser. Alterntively, fctors such s lipoproteins, which re incresed nd ltered in HL-swine ser,2 my ctivte CSF-producing bone mrrow cells in vivo to produce or secrete enhnced levels of M-CSF, block the production of fctors inhibitory to CFU-GM prolifertion, or ct directly on progenitor cells to preferentilly produce monocytes. Experiments tht ddress these questions re now in progress. HL-swine serum contins incresed levels of lipoproteins of ltered composition, most of which belong to the low (LDL), intermedite (IDL), nd very low density (VLDL) lipoprotein frctions.2 In light of the present study, it is interesting tht other investigtors demonstrted n inhibitory effect of lipoproteins on CFU-GM prolifertion in rt, mouse, nd humn bone mrrow cell cultures.1114douy nd coworkers11 demonstrted tht LDL nd high density lipoprotein (HDL3), when dded to bone mrrow cell cultures, mrkedly inhibited CFU-GM prolifertion. In contrst, IDL nd VLDL frctions did not exert ny inhibitory ctivity, nd, furthermore, their ddition to lipoprotein-depleted serum rised progenitor cell prolifertion to the level found in norml serum. Other investigtors, however, found the VLDL frction to be inhibitory to CFU-GM prolifertion,1214 nd Metclf15 indicted tht mouse "light density lipoproteins" suppress grnulocyte colony formtion while committing mouse bone mrrow cells to monocyte formtion in culture. The difference in findings with regrd to the inhibitory effect of VLDL on CFU-GM prolifertion my be ttributed to vritions in the techniques used for the isoltion nd purifiction of lipoproteins, s well s in the culture conditions. If the lipoprotein is isolted under conditions llowing uto-oxidtion or

8 37 Avenil, Megher, nd Germty toxic degrdtion of the lipid prt of the lipoprotein, the inhibitory effect of the lipoprotein my result from lipid peroxide formtion nd ssocited cytotoxic effects.12'1 Helgestd nd coworkers17 showed tht the ddition of reducing gents or free rdicl scvengers to bone mrrow cell cultures enhnced CFU-GM numbers in response to stndrd CSF preprtions. Crude CSF preprtions probbly contin lipoproteins nd other substnces tht directly or indirectly prticipte in the formtion of free rdicls. In our swine BMC culture system, ny potentil deleterious effect of incresed levels of oxidized lipoproteins in HL-swine ser my hve been llevited by the ddition of 2-mercptoethnol, reducing gent. The results of our experiments demonstrted tht HLswine ser elicits enhnced monocytic colony formtion in N-swine s well s in HL-swine BMC cultures. Thus, prior ctivtion or ltertion of BMC in hyperlipemic environment is not necessry for enhnced monocytic progenitor cell prolifertion in vitro in response to fctors in HL-swine ser. However, the results lso demonstrte tht BMC from HL-swine possess n enhnced intrinsic cpbility to proliferte nd form monocytic colonies compred with N-swine BMC, regrdless of whether the stimulus is HL- or N-swine ser. Tken together, these results indicte first, tht incresed BMC prolifertion resulting in the observed monocytosis my not require long-term prectivtion of monocytic progenitor cells by fctors in the hyperlipemic environment. However, once ctivted, BMC retin their incresed cpcity to generte elevted numbers of monocytic progenitor cells. In other words, residence of bone mrrow stem cells within the hyperlipemic environment led to significnt increse in the proportion, sensitivity, or both of monocytic progenitor cells to proliferte in response to circulting in vivo fctor(s). This chnge is not ltered fter removl of BMC from the hyperlipemic environment. Becuse in the current study the enhncing effect of HL-swine serum ws seen in ser obtined from nimls fed the diet for severl months, the minimum time of hyperlipemi required for induction of enhnced cpbility for monocytic colony prolifertion in BMC is unknown. However, monocytosis is evident in these nimls s erly s 1 to 12 weeks fter initition of the diet. It would be interesting to determine in future experiments if there is chronologicl reltionship between initition of monocytosis in HL-swine nd the cpcity of HL-swine ser to induce enhnced numbers of proliferting monocytic colonies in BMC cultures. The results lso hve implictions for plque regression, in tht, if incresed cpcity for monocyte production is retined fter removl of hyperlipemic conditions, persisting monocytosis could significntly contribute to removl of lipid from the plque, s previously postulted.2 In fct, the presence of mny monocyte/mcrophges in regressing lesions hs previously been documented in the swine model. 18,19 In conclusion, the results of these studies re consistent with our previous findings indicting tht the role of the monocyte in therogenesis is, t lest initilly, protective mechnism to remove lipid from lesion sites. The current studies demonstrte first specific response by the bone mrrow to hyperlipemic conditions, in tht greter numbers of BMC re present in hyperlipemic nimls. Second, these cells show enhnced cpbility to form monocytes when exposed to hyperlipemic serum, which my explin the observed monocytosis in these nimls. This process, therefore, provides ugmented numbers of circulting monocytes to prticipte in lipid removl. References 1. Gerrity RG, Richrdson M, Somer JB, Bell FP, Schwrtz CJ: Endothelil cell morphology in res of in vivo Evns blue uptke in the ort of young pigs. II. Ultrstructure of the intim in res of differing permebility to proteins. Am J Pthol 1977, 89: Gerrity RG, Nito HK, Richrdson M, Schwrtz CJ: Dietry induced therogenesis in swine: Morphology of the intim in prelesion stges. Am J Pthol 1979, 95: Gerrity RG: The role of the monocyte in therogenesis. I. Trnsition of blood-borne monocytes into fom cells in ftty lesions. Am J Pthol 1981,13: Gerrity RG: The role of the monocyte in therogenesis. Il. Migrtion of fom cells from therosclerotic lesions. Am J Pthol 1981,13: Gerrity RG, Goss J, Soby L: Control of monocyte recruitment by chemotctic fctor(s) in lesion-prone res of swine ort. Arteriosclerosis 1985, 5:55-5. Gerrity RG: Monocyte-mcrophge recruitment in therogenesis Proceedings of Symposium t the Occsion of the th Anniversry of the University of Heidelberg. Edited by CJ Schwrtz, RM Nerem. New York, Springer-Verlg 1988 (in press) 7. Metclf D, Moore MS: Techniques in experimentl hemtology nd their limittions, Hemopoietic Cells: Frontiers of Biology. Edited by A Neuberger, LL Ttum. North Hollnd, Amsterdm 1971, pp Metclf D: Trnsformtion of grnulocytes to mcrophges in bone mrrow colonies. J Cell Physiol 1971, 77: McNiece IK, Willims NT, Johnson GR, Kriegler AB, Brdley TR, Hodgson GS: Genertion of murine hemtopoietic precursor cells from mcrophge high-prolifertive potentil colony-forming cells. Exp Hemtol 1987,15: Metclf D: Neutrophil nd mcrophge colony formtion by norml cells. Recent Results Cncer Res 1977, 1: Douy L, Brbu V, Billou C, Njmn A, Gorin N, Polonovski J, Duhmel G: The role of serum lipoproteins on the in vitro prolifertive potentil of humn hemtopoietic progenitors CFUC nd CFUe. Exp Hemtol 1983,11:499-55

9 Bone Mrrow Response to Hyperlipemi Zucker S, Beck G, Yi P, Lysik R, DiStefno J: Rt hemtopoietic cell receptor for very low density lipoprotein (VLDL) growth inhibitor. Exp Hemtol 1981, 9: Metclf D, Russel S: Inhibition by mouse serum of hemtopoietic formtion in vitro. Exp Hemtol 197,4: Zucker S, Lysik R, Chikkpp G, Glucksmn H, Gomez-Reino J, DiStefno J: Very low density lipoprotein hemtopoiesis inhibitor from rt plsm. Exp Hemtol 198, 8: Metclf D: Regultion of mcrophge production. Adv Exp Med Biol 1982,155: Chisolm G, Hessler J, Morel D: Low density lipoprotein cytotoxicity induced by free rdicl peroxidtion of lipid. J Lipid Res 1983, 24: Helgestd J, Storm-Mthisen I, Lie S: Endogenous inhibitors of humn grnulocyte/mcrophge colony forming cells in vitro re inctivted by free rdicl scvengers. Scnd J Hemtol 198, 37: Doud AJ, Jrmolych J, Augustyn JM, Fritz KE: Sequentil morphologic studies of regression of dvnced therosclerosis. Arch Pthol Lb Med 1981, 15: Doud AS, Fritz KE, Jrmolych J, Frnk AS. Role of mcrophges in regression of therosclerosis. Ann NY Acd Sci 1985, 454: Acknowledgment The uthors cknowledge Mrs. Ptrici O'Toole for her excellent ssistnce in typing this mnuscript, nd Ms. Jon Kohrs nd Mr. Ken Oliver of the Sigm Chemicl Compny for supplying the esterse stins for testing swine bone mrrow cell cultures. The ssistnce of Dr. Dvid Averill nd Mr. Kirk Esley in sttisticl nlysis of the dt is lso grtefully recognized.