857

Transferrin, Iron, and Serum Lipids Enhance or Inhibit Mycobacterium aviumReplication in Human Macrophages

George s. Douvas, Mary H. May, and Alfred J. Crowle Webb- Waring Lung Institute, Division ofImmunology, and DepartmentofBasic Sciences and Oral Research, University ofColorado School of

Dentistry, University ofColorado Health Sciences Center, Denver

Mycobacterium avium grows exponentially over 7 days in human macrophages when they arecultured in serumless medium. Normal serum inhibits this replication. When serum lipids wereextracted using chloroform, the inhibitor was present in the lipid-free component. The lipidextract significantly enhanced M. avium replication. Iron (Fe2+)added at 8-80 #Lg/mL to infectedmacrophage cultures in serum resulted in enhanced mycobacterial replication. Serum-inducedinhibition of bacterial growth in serumless medium could be duplicated with apotransferrin at50-500 #Lg/mL.At 1000 #Lg/mL, apotransferrin no longer inhibited bacterial growth. Holotrans-ferrin was not inhibitory, and at 500 #Lg/mL, it enhanced M. avium growth. Depletion of thetransferrin in serum by affinity chromatography using goat anti-transferrin on protein G-Seph-arose removed inhibitory activity. These results indicate that transferrin levels, transferrin satura-tion, iron levels, and serum lipids can profoundly alter the replication ofM. avium in associationwith macrophages.

Disseminated infection with Mycobacterium avium is themost common systemic bacterial infection in patients withAIDS [I]. M. aviuni infection is infrequently seen in otherimmunocompromised adults, and before the onset of theAIDS epidemic, M. avium complex infection was only rarelyreported as a significant cause of morbidity or mortality [2].M. aviunt is primarily a parasite of the reticuloendothelialsystem. and infection of patients with AIDS typically in-volves infiltration of the blood, liver. spleen. lymph nodes,and bone marrow [I]. Clinically, disseminated M. avium in-fection presents with high fever. progressive weakness. mal-aise, anemia. and general clinical deterioration [3].

Treatment of M. avium infection with antimicrobials ini-tially showed little promise [4. 5]. More recently. antimicro-bial chemotherapy in patients with AIDS has significantlyincreased their survival [6]. The cost of treatment of M.avium infections is substantial, however. with national esti-mates for drug therapy alone at more than $20 million peryear [7]. The cost for hospitalization and therapy could con-servatively exceed $100 million per year [7]. Understandinghow M. avium replicates intracellularly and why M. avium

Received 13 July 1992: revised 27 October 1992.Informed consent was obtained from all cell and serum donors. Human

experimentation guidelines of the US Department of Health and HumanServices and of the Human Subjects Committee of the University of Colo-rado Health Sciences Center were followed.

Grant support: National Institutes of Health (AI-298l 0).Reprints or correspondence: Dr. George S. Douvas, Department of Basic

Sciences and Oral Research, University of Colorado School of Dentistry,University ofColorado Health Sciences Center. C-286, 4200 E. Ninth Ave.,Denver, CO 80262.The Journal of Infectious Diseases 1993;167:857-64 1993 by The University of Chicago. All rights reserved.0022-1899/93/6704-00 I0$0 1.00

infection is rare in human immunodeficiency virus(HIV)-negative individuals takes on increasing importanceas the AIDS epidemic continues.

Jtudies from this [8] and other laboratories [9. 10] haveindicated that serum from HIV-negative individuals inhibitsthe replication of AIDS-derived M. avium in normal macro-phages. Inhibition is transient, with an initial mycobacterio-static phase followed by a rapid phase of bacterial replica-tion. Escape from the serum-induced inhibition, whichresults in the second phase of growth. may involve transi-tions in the infected macrophages or the bacteria [I I]. Theloss of the serum inhibitor effect begins within 24 h afterinfection and is complete by 48 h.

Sera from AIDS and non-AIDS patients with M. avium-Mycobacterium intracellulare infection had decreased abilityto inhibit macrophage-associated mycobacterial growth [12].Macrophages from these patients were also abnormally per-missive for M. avium, possibly the result of their inability torespond to inhibitor. Others have found no differences in theability of M. avium to grow in macrophages from AIDS pa-tients or HIV-negative individuals [13, 14]. These latter stud-ies. however. used antimicrobials in their cultures, whichmay have slowed bacterial replication and thus may haveinterfered with the ability to see mycobacterial growth.

Investigators have reported factors in serum that inhibitthe extracellular replication of Mycobacterium tuberculosis[15. 16], M. avium. Mycobacterium paratuberculosis [17],and Clostridium welchii [18]. Recently, lactoferrin has beendemonstrated to inhibit intracellular multiplication of Le-gionella pneumophila [19]. and chloroquine has been shownto inhibit its intracellular replication by decreasing iron avail-ability [20]. We examined factors in serum from HIV-nega-tive individuals that affect the intracellular replication of M.avium in normal human macrophages.

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858 Oouvas et al. JIO 1993;167 (April)

Materials and Methods

Bacteria. The preparation of M. avium serovar 4, strain7497, has been described [8, 21]. A smooth transparent colonyof 7497, derived from an AIDS patient, was grown to a densityof 108/mL in Middlebrook 7H9 broth (Difco, Detroit). Aliquotsof this suspension (0.3 mL each) were frozen at -90C untilused.

Macrophage isolation and culture. Mononuclear phagocyteswere isolated and cultured as detailed [22]. Peripheral blood wasobtained from 5 HIV-negative donors (3 white men, I blackman, I white woman) ranging in age from 31 to 59 years. Do-nor's age, sex, and race did not affect the results obtained. Fiftymilliliters of blood was obtained by venipuncture and heparin-ized. The blood was centrifuged on ficoll-hypaque, and themonocytic cells obtained were washed three times and sus-pended at 107/mL in RPMI (GIBCO Laboratories, Grand Is-land, NY) supplemented with I% unheated AB serum. Thestock supply of AB serum used throughout these experimentswas obtained from a white, HIV-negative, 53-year-old man.

Cells were plated for 30 min at 37C, 7.5% CO 2 in air, in35-mm bacteriologic grade plastic Petri dishes (Falcon 1008;Becton Dickinson Labware, Oxnard, CA). Nonadherent cellswere washed from the dishes using warmed RPMI withoutserum. The adherent cells were incubated for 6 days in 1.5 mLof RPMI containing 20 mM L-glutamine and I% unheated ABserum. On day 6, the culture medium was removed and replacedwith RPMI containing 1% of a serum substitute (SS). The SScontained 40 mg/rnl, fatty acid-free serum albumin (Sigma, St.Louis), 0.8 mg/rnl. t-o-phosphatidylcholine (Sigma), 10 JLg/mLhuman apotransferrin (Sigma), and 0.2 mg/rnl. cholesterol(Sigma) in bicarbonate-free Iscove medium (GIBCO) [23). The7-day-old macrophage cultures contained r-: 1.5 X 105 macro-phages/Petri dish.

M. avium infection ofmacrophage cultures. For infection ofmacrophage cultures, frozen 0.3-mL aliquots of M. avium sus-pension were thawed rapidly in a 37C water bath. The suspen-sion was then diluted to 3.0 mL with RPMI tissue culture me-dium. This was then ultrasonicated for lOs to disperse thebacteria [21, 24]. M. avium suspensions were diluted to 5 X105/ mL in RPMI supplemented with 1% unheated AB serum.After removal of the culture medium from the macrophages, 1.5mL of the bacterial suspension was added to each plate, and theplates were incubated at 37C, 7.5% CO 2 , for 60 min. The infec-tion medium was then removed, and the plates were washedthree times with 1 mL of warmed RPMI to remove extracellularbacteria. This procedure resulted in ,......, 10% of the macrophagesinfected, with 1-2 bacteria/cell [25]. Next, 1.5 mL of RPMI(containing 1% SS) and 20 mM L-glutamine, with or withoutadditions, were added back to the infected cultures. These wereincubated at 37C, 7.5% CO2 , until harvested.

Determination ofcolony-forming units. Samples were takenimmediately after infection (time 0) and 4 and 7 days after infec-tion for colony-forming unit (cfu) counts. The supernatantswere removed and saved, and the macrophages from each platewere lysed using 1.5 mL of a lysing solution containing 1.1 mLof7H9 medium and 0.4 mL of 0.25% SDS (in physiologic phos-phate buffer) [24]. The Iysates and supernatants from each platewere combined, and 2 mL of this was transferred to a 5-mL tube

containing 20% bovine serum albumin (Sigma) to neutralize theSDS. Each tube was then ultrasonicated for lOs to disperse thebacteria, and 0.2-mL volumes ofthe lysates were serially dilutedin 7H9 medium. Four or five 15-ttL inocula per dilution werethen plated on 7H I0 agar (Difco).

The 7H 10 plates were placed in plastic bags (Micro-Seal; Da-zey, Kansas City, MO), sealed, and incubated for I week at37C in a humidified atmosphere. Each sample time usuallyrepresents duplicate macrophage culture plates. Thus, each re-ported cfu value represents the mean of 8-10 spots. One milli-liter of lysate was the product of r-- 105 lysed macrophages. Eachexperimental group was observed by phase microscopy for verifi-cation of cell culture health. The numbers and characteristics ofthe macrophages in the various experimental groups were com-parable. Results are reported as means SE. Groups were com-pared statistically using Student's t test.

Transferrin and iron sources. Apotransferrin ("""'95% ironpoor) and holotransferrin (98% iron saturated) were obtainedfrom Sigma. Ferrous ammonium sulfate was used as an exoge-nous iron source. Ferrous ammonium sulfate and ferric ammo-nium citrate were compared in several of these experiments,with equivalent results.

Lipid extraction of human serum. Serum was initially frac-tionated into lipid and nonlipid components using methanol-chloroform extraction as described [26]. First, 2.5-mL equiva-lents of lyophilized human serum (Sigma) were suspended in7.5 mL of methanol-chloroform (2: 1). This was shaken inter-mittently for 1-2 h, then centrifuged at 500 g for 10 min. Thesupernatant was saved, and the pellet was suspended in another7.5 mL of methanol-chloroform. After 1-2 h, the contents wereagain centrifuged at 500 g for 10 min. The pellet and superna-tant were then air dried and resuspended in 2.5 mL of distilledwater.

Subsequent extractions were done by suspending a 5-mLequivalent of lyophilized serum into 10 mL of chloroform. Thiswas shaken intermittently for 1 h. The chloroform layer (lower)was removed by pipetting, and the chloroform-insoluble compo-nent was resuspended in another 10 mL ofchloroform. This wasagain shaken intermittently for 1 h. The chloroform layer wasremoved, and the serum was extracted a third time. The chloro-form-insoluble and -soluble components were then air dried andresuspended in 5 mL of distilled water. This latter procedureresulted in less protein denaturation than the methanol-chloro-form extraction procedure. Extraction routinely resulted in>96% reduction oftriglycerides and cholesterol in the extractedfraction as determined by clinical laboratory analysis. There wasusually >90% recovery of triglycerides and cholesterol in thechloroform-soluble component.

Protein G-Sepharose-anti-transftrrin fractionation of normalserum. Goat anti-human transferrin fractionated antiserum(Sigma) was coupled onto protein G-Sepharose as described[27]. Protein G insolubilized on Sepharose 4B (G-Seph), with anantibody binding capacity of 20 mg/ml., was obtained fromSigma.

Before antibody adsorption, 3 mL of G-Seph was washedtwice with unsupplemented RPMI. For washing, the G-Sephwas placed in a conical centrifuge tube and centrifuged at 500 gfor 10 min. The supernatant was removed, and 10 mL of un sup-

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1ID 1993; 167 (April) Modulators of M. avium Growth 859

047Days After Infection

Figure 1. Methanol-chloroform-soluble fraction from ABserum enhances whereas insoluble fraction inhibits M. avium repli-cation. Seven-day macrophages were infected and cultured inRPMI containing I%serum substitute (SS) or 1% SS with 10% ABserum (AB), 10% methanol-chloroform extracted serum (EAB), or10% methanol-chloroform lipid extract (LAB). Infected macro-phages were lysed immediately after infection or at 4 and 7 days,and colony-forming units (CFU) per plate were determined as de-scribed. Bars represent SE. *, P < .0 I compared with SS controls.

form-soluble component enhanced M. avium replication.Comparable results were obtained in at least five other ex-periments using chloroform or ether extraction of serum.

Effect ofiron supplementation on macrophage-associated M.avium replication. Further fractionation of serum indicatedthat the serum inhibitor was present in the supernatant afterfractionation of serum with 33% saturated ammonium sul-fate. The inhibitory activity of the supernatant was trypsin-sensitive, indicating that the serum inhibitor was a protein.Conversely, a lipid-containing component in serum was pre-cipitable by ammonium sulfate and insensitive to trypsiniza-tion and could enhance M. avium replication. The propertiesof the serum inhibitor were consistent with those of trans-ferrin [30].

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Results

Lipid extraction ofAB serum. Initial efforts to biochemi-cally characterize the factors in serum that inhibit M. aviumreplication used the fractionation of normal AB serum intotriglyceride- and cholesterol-depleted and lipid-rich fractionsby methanol-chloroform (2: 1), chloroform, or ether extrac-tions. Results shown in figure I obtained with methanol-chloroform extraction indicate that the inhibitory activitywas retained in the methanol-chloroform-insoluble portionof serum. Analysis of the whole lyophilized serum used infigure I and the serum fractions indicated that the serumcontained 127 mg/dl. cholesterol (normal range, 126-233)and 60 mg/dL triglycerides (normal range, 30-140). Themethanol-chloroform-soluble component contained 116mg/dL cholesterol and 55 mg/dL triglycerides, while the ex-tracted serum fraction contained 4 mg/dL cholesterol andundetectable levels of triglycerides. The methanol-chloro-

plemented RPMI was added. The G-Seph was gently resus-pended by pipetting. This was once again pelleted by centrifuga-tion. The procedure was repeated once more, and after thesupernatant was removed, 2 mL ofanti-transferrin antibody wasadded to the G-Seph. This was reacted for 2.5 h at 4C withoccasional agitation. The G-Seph was pelleted by centrifuga-tion, the uncoupled antibody was removed, and the Sepharosewas washed twice with 10 mL per wash, using RPMI.

After the last wash, 2 mL of RPMI was added to the G-Seph,and one-third of the Sepharose was then placed into a 1.5-mLpolypropylene microcentrifuge tube (VWR Scientific, San Fran-cisco) with a hole punctured in the tip using a 20-gauge needle.Over the hole was placed --2 mm of autoclaved glass wool toprevent the G-Seph from running through the hole. The micro-fuge tube was then fitted snugly into place on the top of a 5-mLcryotube (Nunc; Inter Med, Roskilde, Denmark) that had a holeplaced -- I em from the rim of the tube. The G-Seph in themicrofuge tube, which in turn was in the cryotube, was centri-fuged at 500 g for 10 min to remove excess RPMI.

The anti-transferrin-coupled G-Seph was then resuspendedwith 1.5 mL of AB serum and placed into an unaltered 1.5-mLmicrocentrifuge tube. This was incubated for 2.5 h at 4C, withoccasional agitation. After the absorption time, the mixture wasplaced into a microcentrifuge tube with a hole in its bottomcovered with glass wool, and the absorbed AB serum was centri-fuged into the 5-mL cryotube as before. The AB serum was onceagain reacted with the second volume of anti-transferrin-cou-pled G-Seph for 2.5 h as before. The AB serum was centrifugedout and absorbed for a third time with the third batch ofG-Seph.This procedure yielded --80%-85% recovery ofthe absorbed ABserum.

Depletion of transferrin from AB serum by the above methodwas monitored using the single radial immunodiffusion tem-plate test as described [28, 29]. Transferrin levels were deter-mined by comparison with standard concentrations ofapotrans-ferrin. Holotransferrin and apotransferrin standards gaveequivalent results. The limit of sensitivity of this assay was --10JLg/mL transferrin.

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Figure 3. Apotransferrin inhibits whereas ho1otransferrin canenhance M . avium replication. Seven-day maerophages were in-fected with M . avium and cultured in RPMI containing 1% serumsubstitute (SS), RPMI containing 1%SS with 10%AB serum (AB),or 5-500 ~gjmL apotransferrin (APO) or holotransferrin (HOlO).Immediatelyafter infection (0 time) or at 7 days. macrophageswerelysed and colony-forming units (CFU) determined. Values aremeans + SE. *, P < .0 I compared with SS controls.

induced inhibition and, at concentrations >8 JLg/mL, en-hanced M . avium growth.

Effects ojapotransferrin and holotransferrin on mycobacte-rial replication. Results from the above experiments con-firmed the importance of iron in mycobacterial replicationand further strengthened the possibility that the serum inhibi-tor was an iron -binding protein such as transferrin . We nextexamined directly whether purified transferrin, either iron-free (apo-) or iron -saturated (holo-) , cou ld affect M. aviumgrowth. Figure 3 represen ts results from one of four experi-ments that indicate that 50-500 JLg/mLapotransferrin couldinhibit macrophage-associated M. avium growth; 5 and 50JLg/mL holotransferrin were ineffective at inhibiting M.avium growth, and 500 JLg/ m L holotransferrin enhanced my-cobacterial rep lication .

Effect oj iron supplementation on apotransferrin-inducedinhibition. Iron supplementation not only eliminated ABserum-induced inhibition of M . avium growth but, at ~8JLg/mL, could enhance growth (Figure 2), and transferrinmay be the serum inhibitor (Figure 3) . We next examined

Douvas et al.

3 ""'---.........'----"_Figure 2. Iron supplementation eliminates serum-induced inhi-bition. Seven-day macrophage cultures were infected with M .avium; immediately thereafter, RPMI containing I%serum substi-tute (SS), 1%SS and 0.8-80.0 ~g/mL ferrous ammonium sulfate(Fe), 1%SS and 10%AB serum (AB), or 1%SS with 10%AB and0.8-80.0 ~gjmL Fe was added . Cultures were lysed, and colony-forming units (CFU) were determined at 0 time or 7 days afterinfection. Values are means + SE. *. P < .0 I compared with SScontrols .

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Because of the importance ofiron in mycobacterial growthand the association of transferrin-like properties with theserum inhibitor. we next examined whether iron supplemen-tation affected intramacrophage mycobacterial growth. Fig-ure 2 shows that addition of ferrous ammonium sulfate inconcentrations ranging from 0.8 to 80.0 JLg/mL had littleeffect on M. avium growth in SS alone. Several other experi-ments have indicated that iron supplementation alone, in SS,can slightly enhance mycobacterial replication over SS-con-taining medium. Addition of~8 JLg/mL iron in the pre senceof AB serum enhanced mycobacterial rep lication. Theseconcentrations of iron are in excess of concentrations re-quired to saturate the transferrin [3l] . Ferrous ammoniumsulfate and ferric ammonium citrate were compared in sev-eral experiments wit h equivalent results. Ferric ammoniumcitrate, however, was slightly more effective in enhancingmycobacterial growth in the presence of AB serum (data notshown). Thus, iron supplementation could eliminate serum-

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JID 1993;167 (April) Modulators of M. avium Growth 861

whether iron could eliminate transferrin-induced inhibition,similar to its ability to do so with whole serum. Figure 4represents results from five experiments that demonstratethat 80 Jtg/mL iron eliminated the transferrin-induced inhibi-tion . In a number of experiments, iron supplementation notonly eliminated transferrin induced inhibition but slightlyenhanced M. avium growth over SS controls.

Affinity column depletion of normal serum. Biochemicalcharacterization of the serum inhibitory component sug-gested that it had properties similar to transferrin . Severalother experiments indicated that purified transferrin couldact as the inhibitor. Definitive proof that transferrin was theinhibitor in human serum could best be obtained by deplet-ing transferrin from whole serum and examining the activityof the transferrin-depleted serum. To do this. goat antibodiesagainst human transferrin were adsorbed onto protein G-Sepharose. The protein G-Sepharose-anti-transferrin com-plex was then used to deplete transferrin from AB serum.Transferrin levels in the absorbed AB serum, as determined

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Figure 4. Iron eliminates transferrin-induced suppression of M.avium growth. Seven-day macrophages were infected with M.avium and cultured in RPMl containing I%serum substitute (SS).or 1%SS with 10% All serum (AB), 80 Jlg/mL ferrous ammoniumsulfate (Fe). 50-100 Jlg/mL apotransferrin (Tr), or 50-100 Jlg/mLTr and 80 Jlg/mL Fe. Macrophage cultures were lysedat 0 time or 7days after infection for colony-forming unit (CFU) determination.Barsrepresent SE. e , P < .0 I compared with SScontrols. , P < .0 Icompared with Tr controls.

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Figure 5. Depletion of transferrin from AB serum by affinitychromatography removes serum-induced inhibition of M . aviumreplication: 10% transferrin-depleted AB serum in I%serum substi-tute (TD AB) was compared with 1%-10% whole AB serum inserum substitute (AB) or serum substitute alone (SS). Cell cultureswere harvested immediately after infection (0 time) or at 7 days.Valuesare means + SE. *. P < .01 compared with SS controls.

by single radial immunodiffusion. were < I0 Jtg/mL. Figure 5shows results from one of three experiments indicating thatdepletion of transferrin from serum resulted in the abroga-tion of inhibition. and in fact transferrin-depleted serum ef-fectively stimulated M. avium replication. Enhanced myco-bacterial replication is most likely explained by thecontribution of the serum lipid component (see figure I) .Mock absorption of AB serum on protein Ge-Sepharose hadno effect on the inhibitory activity of serum (data notshown).

Lack ofincreased suppression ofmycobacterial growth withhigh levels oftransferrin. The ultimate goal of these experi-ments was to identify the serum inhibitor. It was then antici-pated that the purified inhibitor could be used to controlmacrophage-associated M. avium replication. Previous ex-periments indicated that inhibition of bacterial replication

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862 Douvas et al. JID 1993;167 (April)

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Figure 6. High concentrations oftransferrin do not inhibit intra-macrophage M. avium replication. Seven-day macrophages wereinfected with M. avium and cultured in RPMI containing I%serumsubstitute (SS), 1% SS and 10% AB serum (A B), or 1%SS with 50,500, or 5000 ~g/mL apotransferrin (Tr). Infected macrophageswere lysed immediately after infection or at 4 and 7 days for deter-mination of colony-forming units (CFU). Results are means SE.*, P < .0 I compared with SScontrols. -, P < .05 compared with SScontrols.

could not be improved by using concentrations of serum> 10%-20% (data not shown). However, the above experi-ments demonstrated that serum also contained lipid compo-nents that could enhance replication. Thus, it was postulatedthat the reason bacteriostasis or bactericidal activity couldnot be obtained at higher serum concentrations was that theenhancing components in serum overrode the inhibitor athigh serum concentrations. Having identified the inhibitor astransferrin, we next examined whether high levels of trans-ferrin, similar to those found in undiluted serum, could in-crease mycobacterial growth inhibition. The results from oneof four experiments are shown in figure 6, which illustratesthat at high concentrations ofapotransferrin (5000 p.g/mL),growth inhibition actually decreased. Thus, transferrin aloneat serum concentrations cannot significantly inhibit M.avium replication.

Discussion

Serum from HIV-negative individuals inhibits the replica-tion of M. avium in cultured human macrophages [8, 10].The inhibition is transient [8, 11], with an initial slow periodof growth for the first 4 days in culture, followed by rapidmycobacterial replication from 4 through 7 days. Inhibitordelays the transition of macrophages or mycobacteria to thestate that results in rapid mycobacterial replication [11]. Thetransition of the macrophages or mycobacteria becomes evi-dent within the first 24 h after infection. Once the transitionhas occurred, mycobacterial growth is no longer inhibited.

Initial serum fractionation using ammonium sulfate precip-itation and methanol-chloroform extraction (figure 1) pro-vided results consistent with the possibility that the seruminhibitor was transferrin [30]. This hypothesis was strength-ened by results obtained using ion exchange chromatogra-phy and PAGE (data not shown). Definitive proof that theserum inhibitor was transferrin was obtained by the demon-stration that purified transferrin had the same activity asserum (figures 3,4,6) and that transferrin depletion of nor-mal serum by affinity chromatography removed inhibitoryactivity.

Transferrin and lactoferrin have been shown to inhibit mi-crobial growth in a number of other systems. Kochan andcolleagues [15, 31] demonstrated that transferrin inhibitedthe growth of M. tuberculosis in a cell-free system. Like ourgroup, they found that the higher the degree of iron satura-tion ofthe transferrin, the less the inhibition. Transferrin wasalso shown to inhibit the growth of Histoplasma capsulatum[32]. In culture and in a mouse model, the addition of ironeither neutralized the inhibition of H. capsulatum growth[31, 32] or exacerbated the course ofinfection [33]. Byrd andHorwitz [20] also demonstrated the necessity for iron in L.pneumophila growth. Apolactoferrin inhibited the intracellu-lar replication ofL. pneumophila [19, 20], whereas iron-satu-rated lactoferrin had no effect on the intracellular replicationrate.

The addition of iron to infected macrophages cultured inwhole serum (figure 2), and in some instances purified trans-ferrin, enhanced M. avium growth over SS controls. Growthenhancement was also seen by the addition of iron-saturatedtransferrin (figure 3). Iron could induce increases in trans-ferrin receptor expression by macrophages, which would re-sult in increased intracellular iron stores. Testa et al. [34]reported that the addition ofiron to cultured monocytes doesenhance transferrin receptor expression. Sciot et al. [35] de-scribed an increase in the expression of transferrin receptorson Kupffer cells in the presence of iron. Jandl et at. [36] hadpreviously demonstrated that iron uptake from transferrin byliver slices increased with increased transferrin saturation.More recently, White and Jacobs [37] obtained similar re-sults with Chang liver cells, and Worrall and Worwood [38]demonstrated that incubation ofmonocytes with iron caused

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JID 1993;167 (April) Modulators of M. avium Growth 863

an increase in monocyte ferritin levels. Thus, increasing ei-ther the transferrin saturation or the concentration of ironcould induce macrophages to provide essential iron thatwould enhance M. avium replication.

Serum iron levels and transferrin saturation levels in pa-tients with AIDS are low (transferrin levels are normal) [39,40]. However, iron storage in cells such as macrophages isincreased in AIDS, as in other chronic diseases [41, 42]. Thecoexistence of iron and mycobacteria in macrophages, thecells most commonly parasitized by M. avium, may lead toaccelerated mycobacterial replication in infected cells andmay predispose AIDS patients to overwhelming mycobacte-rial replication.

Iron supplementation in these experiments was done us-ing ferrous (Fe2+) ammonium sulfate. Many of the experi-ments were replicated using ferric (Fe3+) ammonium citratewith similar results. Although iron in the transferrin-ironcomplex is in the ferric state [3o], transferrin at neutral pHwill react readily with Fe2+ [30]. It is postulated that Fe2+may attach to the specific binding site for iron on the trans-ferrin and is rapidly oxidized to the trivalent form.

Figure I shows that a third component important in theregulation ofM. avium growth by serum is soluble in metha-nol-chloroform or chloroform. Laboratory analysis has dem-onstrated that the chloroform-soluble fraction is rich inserum cholesterol and triglycerides. This fraction may con-tain a number of nonlipid contaminants such as sugars,ureas, salts, and amino acids [43], as well as some carryoverprotein contaminants. Trypsinization of this fraction did noteliminate its growth-enhancing capabilities (data notshown), suggesting that the serum enhancer is not a protein.Components in the lipid-rich fraction, such as triglycerides,may act as growth promoters. Triglycerides have been shownpreviously to potentiate mycobacterial growth [44], and hy-pertriglyceridemia has been reported as a common occur-rence in patients with AIDS [45]. Additionally, chloroform-soluble fatty acids similar to oleate or palmitate, which areessential for M. avium growth [46], could playa role in theincreased mycobacterial replication. Further biochemicalcharacterization of this fraction is underway.

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