Human Alveolar Macrophage-derived
Chemotactic Factor for Neutrophils: STIMULI
AND PARTIAL CHARACTERIZATION
Gary W. Hunninghake, … , Henry M. Fales, Ronald G.
J Clin Invest.
The presence of neutrophils within the lung is a characteristic feature of a variety of lung
diseases. To evaluate the potential role of alveolar macrophages in modulating the
migration of neutrophils to the lung, normal human alveolar macrophages obtained from
volunteers by bronchopulmonary lavage, were exposed for various periods of time in vitro to
heat-killed microorganisms, and noninfectious particulates, immune complexes, and the
macrophage supernates were evaluated for chemotactic activity. The microorganisms,
noninfectious particulates, and immune complexes were chosen as stimuli for alveolar
macrophages because these stimuli are representative of a spectrum of pathogenic agents
that cause neutrophil accumulation in the lower respiratory tract. After incubation with each
of these stimuli, alveolar macrophages released low molecular weight (400-600)
chemotactic factor(s) (alveolar macrophage-derived chemotactic factor[s] [AMCF]) with
relatively more activity for neutrophils than monocytes or eosinophils. Checker-board
analysis of the AMCF revealed that the factor was primarily chemotactic and not
chemokinetic for neutrophils. The selectivity for neutrophils vs. monocytes could not be
explained by a selective deactivation of monocytes, because the AMCF was more potent in
deactivating neutrophils than monocytes. Partial characterization of AMCF demonstrated it
was heterogeneous with the following features: (
) stable to heating at 56 and 100°C for 30
) stable over a pH range of 1.0 to 12.0 for 60 min; (
) stable after exposure to […]
Alveolar Macrophage-derived Chemotactic
GARY W. HUNNINGHAKE, JAMES E. GADEK, HENRY M. FALES, and RONALD G.CRYSTAL,
Pulmonary Branch and Laboratory of Chemistry, NationalHeart, Lungand BloodInstitute,National Institutesof Health, Bethesda, Maryland20205
S T R A C T The presence of neutrophils within the
a characteristic feature of a variety of lungdis-eases. To
evaluate the potential role of alveolar
modulating the migration of neutrophils to
the lung, normal human alveolar macrophages obtained
from volunteers by bronchopulmonary lavage, were
periods oftimeinvitro to
microorganisms, and noninfectious particulates,
immune complexes, and the macrophage supernates
evaluated for chemotactic activity. The
micro-organisms, noninfectious particulates, and immune
complexes were chosenas
macro-phages because these stimuli are representative of a
the lower respiratorytract.
incu-bation with each ofthese stimuli, alveolar
released low molecular weight (400-600) chemotactic
neutrophils than monocytesor
analysis of theAMCF
revealed that the factorwas
primarily chemotactic andnot
neutrophils. The selectivity for neutrophilsvs.
of monocytes, because theAMCF was more
deactivating neutrophils than monocytes. Partial
heterogeneous with the following features: (a) stable to
heating at 56 and 100°C for30
(b) stable over a pH
min; (c) stable
trypsin, papain, chymotrypsin, collagenase, and
elastase; (d) partiallyinhibited
This paperwaspresentedin partatthe NationalMeeting
oftheAmerican Federationfor ClinicalResearch, San Fran-cisco, Calif.,May 1978.
Receivedfor publication13September1978and in revised
and 5.2); and (f) partially extractableinto
acid); analysis by
reveal the presenceof5,8,10,14-eicosatetra-enoic
release lysozyme and lactoferrin butnot
dehy-drogenase. These studies suggest thata
stimuli induce normal
residents of alveoli,it is
factor(s) macrophages playa
inflammatory processesseen in
chronic lung diseases.
has been well documented that an importantfunc-tion
of the alveolar macrophageis
of the lower respiratory tract by ingesting
agents (1, 2).Recent
models have suggested that the
respiratory tract by secreting chemotactic factor(s)
other types of inflammatory andimmune
cells, particularly neutrophils,to
(3, 4). Such alveolar macrophage-derived
im-IAbbreviations usedinthispaper:AMCF, alveolar
macro-phage-derived chemotactic factors; BSA, bovine serum
portanceinthehumanlung,notonlybecauseofitsrole in lung defense, but also because the production of AMCF may help explain the accumulation of neutro-philsinalveolarstructures in association withavariety
If the release
of suchchemotactic factor(s) for
their production shouldbe associated with the interac-tion
of the alveolar macrophagewith those
pathogenicagents known to cause neutrophil accumulation in the lower respiratory tract (5-14). Since neutrophils appear in the
lung within hours ofa
expectedthat AMCF would be released
followinginteraction of the macrophage witha
poten-tiallyinjurious agent. In
addition, becauseneutro-phils are rarely found in the alveolar structures of
normal lung,it is
unlikelythat the resting,
unstimu-lated alveolar macrophage would releaseAMCF.
evaluatethe potential role
modulation of neutrophilmigration to the
have examinedthe capacity of various pathogenic agents to stimulate
nor-mal human alveolar macrophagesto generate and
re-lease chemotactic factor(s) for neutrophils. Thiswas
accomplished by obtaining alveolar macrophages from
normal volunteers by bronchoalveolar lavage,cul-turing
macrophages with different classes of
pathogenic stimuli, andtesting
for neutrophilchemotactic activity.
Preparation of blood leukocytes. Mononuclear cells and neutrophils were obtained from peripheral blood of normal volunteers by sedimentation in pyrogen-free Plasmagel (HTI Corp., Buffalo, N. Y.) followed by Hypaque-Ficoll centrifugation (15). The resulting mononuclear cell suspen-sions contained >98% mononuclear cells of which -80% werelymphocytes and20%weremonocytes, while the poly-morphonuclear leukocytes (PMN) suspensions contained
>98% neutrophils. In those studies designed to evaluate the relative selectivity of the macrophage chemotactic
fac-tor for neutrophils compared with eosinophils, PMN
sus-pensions were also obtained from three individuals with peripheralbloodeosinophiliaandallergic rhinitis secondary
to a documented ragweed allergy. PMN suspensions from these three individuals contained 8-20% eosinophils and
Bronchoalveolar lavage. Bronchoalveolar lavage was performedon sixnonsmoking,healthy volunteers(mean age
21±1yr) aspreviously described (7,13, 14). The cells were
separated from lavage fluid by centrifugation, washedthree times, and then evaluated for total number, viability, and
differentialcount (14).(Alldata arepresentedas mean+SEM). Preparation of alveolar macrophages. In the six
volun-teers theaverage lavage fluid cell differential of these
indi-viduals included: 92±1% alveolar macrophages, 7+1% lymphocytes,and 1+1%neutrophils; eosinophils, basophils,
and mast cells were not found. Viable mononuclear cells
were further purified from the recovered cells by Hypaque-Ficoll densitycentrifugation;theresulting mononuclear cell
suspensions were >98%viable and contained,ontheaverage, 93% macrophages and7% lymphocytes. These cell suspen-sions(referredtosubsequentlyas"macrophages") werethen adjusted to aconcentration of107/mlin RPMI-1640
(Micro-biological Associates, Walkersville, Md.) and were usedto generatethe macrophagesupemates,as describedbelow.
Classes of materials used to stimulate alveolar
macro-phages. Three classes of materials were used to stimulate
the alveolar macrophages obtained by bronchoalveolar lavage: (a) microorganisms; (b) noninfectious particulates; and (c) immunecomplexes.
Because some living microorganisms produce chemotactic
(16,17), themicroorganisms used inthis study were
heat-killed before use. Heat-killed Staphylococcus albus (National Institutes of Health microbiological service) was
used as an example of a bacterial stimulus and zymosan
(yeast cell walls) particles (Sigma Chemical Co., St. Louis, Mo.) wereusedas an example ofa nonbacterial, particulate infectious agent.TheS. albuswere adjusted to a concentra-tion of 5x 108 organisms/ml in RPMI-1640. The zymosan
particles were prepared as described by Fine et al. (18), washed threetimes inHanks'balancedsaltsolution (HBSS), andresuspendedinRPMI-1640to a concentrationof1mg/ml.
As anexampleof anoninfectious particulate, Sepharose-4B particles (Pharmacia Fine Chemicals, Div. of Pharmacia,
Inc., Piscataway, N. J.) wereused after washingtheparticles threetimesinHBSSandresuspending theminRPMI-1640at aconcentrationof10%by volume.
Toevaluate the potential influence ofopsonization onthe ability of the microorganisms or Sepharose-4B to act as
stimuli for the alveolar macrophages, the heat-killedS.
zymosan,andSepharose-4B wereopsonizedbyincubationin fresh normal human serum for1h at 37°Cfollowed by three washings in RPMI-1640and final suspension in RPMI-1640 atthe concentrationsdescribed above.
Bothsolubleand particulate immunecomplexeswere used as examples ofimmune complex stimuli. Soluble
antigen-antibody complexes were prepared from bovine serum
al-bumin (BSA) and immunoglobulin (Ig)G anti-BSA antibody raised in rabbits. Antigen-antibody complexes were
pre-paredat five times antigen-excess in RPMI-1640and
repre-sentedatotalprotein concentrationof1 mg/ml.
Three different particulate immune complexes were
usedaspotential stimuli: (a) oxerythrocytes(ORBC)coated withjustsubagglutinating amountsof rabbitIgGanti-ORBC
antibody(7S EA; N. L. Cappel Laboratories, Inc., Cochran-ville, Pa.) and suspended in RPMI-1640 at a concentration
of5 x 108 cells/ml; (b)ORBCcoatedwith just
subagglutinat-ing amounts of rabbit IgM anti-ORBC antibody (19S EA;
Cappel Laboratories, Inc.) and suspended in RPMI-1640 at aconcentration of 5x 108 cells/ml;and (c)sheep erythrocytes
(SRBC) coated with subagglutinatingamounts of rabbit IgM anti-SRBCantibody andmousecomplement (EAC) and
sus-pendedinRPMI-1640 at 5 x 108cells/ml.
Incubation ofalveolarmacrophageswith various stimuli. Toevaluate theproductionof chemotactic activityby alveolar macrophages,themacrophageswereincubatedinsterile tis-suecultureplates (24 wells/plate, 16 mmwellDiam; Costar, Data Packaging, Cambridge, Mass.) at 37°C in 5% CO2 in airat 100%humidityon arockerplatform (Bellco Glass,Inc.,
Vineland, N. J.) (7 cycles/min) for various periods oftime, eitheralone orinthe presence of oneoftheseveral stimuli describedabove. Themacrophagecultureswerepreparedby
salt solution; HETE, 5,8,10,14-eicosatetraenoic acid; LDH,
lactic acid dehydrogenase; ORBC, ox erythrocytes; PBS,
mixing 0.8 ml ofRPMI-1640, 0.1 ml of the alveolar
macro-phage suspension, and 0.1 ml of the various stimuli in wells of the tissue culture plates. At various times (0, 0.5, 1, 2, 3, 4,
and18h) afterinitiationofthecultures,the macrophage super-nates were harvested by aspiration and filtered through
0.45-gmfilters (Swinnex-13, Millipore Corp., Bedford, Mass.) to remove cells and the particulate stimuli. Control prepara-tionsfor these experiments included supemates from alveolar
macrophages alone and supemates from each of the stimuli
alone.Allsupernates werestoredat-20°C and were assayed within 2 wk of harvesting.
Evaluation of alveolar macrophage supernates for
chemo-tactic activity. Toevaluate the alveolar macrophage super-nates for chemotactic activity directed toward neutrophils ormononuclear cells, ablindwell micropore filter chamber technique was used with nucleopore filters
forpolymorphonuclear leucocytes, 5-,umpore size for
mono-nuclear cells; Neuro Probe, Inc., Bethesda, Md.), as
previ-ously described (19).
To insure that the migration of neutrophils across the membraneinresponse tothemacrophagesupemates wasdue
to directed migration (chemotaxis) and not merely
in-creased random migration (chemokinesis) (20), in some ex-periments, varying amounts of the chemotactic stimulus were added to both the upper and lower compartments of the chamber.
To validate the results of these assays, chemotaxis
was also evaluated using Sartorius filters (Sartorius AG, Gottingen,WestGermany)in aleading frontassay as
previ-ously described (4) and achemotaxis under agarose assay (21). The results obtained using the various chemotactic assays werecomparable.
deacti-vateneutrophilsvs.mononuclear cells, neutrophiland
mono-nuclear cell suspensions were incubated with varying amountsofAMCFbefore theiruse in the chemotaxisassay.
After incubation with AMCF at 37°C for 15 min, the cells
werewashed three times andplaced in the upper compart-ment(cell) of the chemotaxis chamber. Intothe lower com-partment(stimulus) of the chamberwasadded150,ulof either media alone (RPMI-1640), AMCF, N-formyl-Met-Leu-Phe (50 nM; Peninsula Laboratories, Inc., San Carlos, Calif.),
or 5%endotoxin-activatedserum (22). Themigration ofboth neutrophils and mononuclear cellsacrossthe nucleopore
fil-ter in response toeach ofthese stimuli was then evaluated
Tocompare therelative chemotacticactivityof the
macro-phage supernates for neutrophils vs. eosinophils, PMN sus-pensions containing both neutrophils and eosinophils were
preparedasdescribed above.Fortheseexperiments, a chemo-taxis-under-agarose assay (21) was used, in addition to the nucleoporefilter assay.
Partial characterization of AMCF. Molecular sieve
chromatography ofthealveolar macrophage supemates was
performed on a 2.5 x 100-cm column of Sephadex G-25
(PharmaciaFineChemicals) anda 1.5x 90-cmcolumnof
Bio-Gel P-2 (Bio-Rad Laboratories, Richmond, Calif.). The
col-umnswerepackedandelutedwith 0.85%NaCl-40mM phos-phate, pH 7.4phosphate-buffered saline (PBS) at 4°C. 3-ml
fractions werecollectedandassayed directly for chemotactic
activityusing thenucleoporefilter assay. The G-25Sephadex
column was calibrated with cytochrome C (12,500 mol wt), bacitracin (1,450mol wt)and[3H]thymidine(242mol wt). The Bio-GelP-2column was calibrated with vitamin B12 (1,350 mol
Isoelectric focusing of AMCF was performed in a
hori-zontalbedofgranulated gel (Ultrodex;LKBInstruments, Inc., Rockville, Md.).ApHgradientof 2.5-9wasestablishedusing
a composite of ampholytes (Bio-Rad Laboratories) at a final concentration of 2% (wt/wt) in the gel slurry (23). AMCF partially purified by prior gel filtration was added to the granulated gel bed (3% vol/vol) before applications of electro-focusing current. Isoelectric electro-focusing was carried out at 25 W,
5°C, for 16 h. The gel was then collected as 75-mm strips proceeding fromanode tocathode. The pH of each step was determined and then eluted with 3 ml of PBS. The eluate from the gel strips was then assayed for neutrophil chemotactic activity as described above. Control isoelectric focusing was
performed on alveolar macrophagesupemates containing no
chemotactic activity. Analysis of control runs demonstrated that the granulated gel-ampholyte focusingbed itself did not contain measurable chemotacticactivity for neutrophils.
The heat stability of the AMCF was evaluated by heating the
macrophagesupematesfor 30minat 56 and 100°C. The sensi-tivity ofAMCF to trypsin, chymotrypsin, papain, clostridial
collagenase, and pancreatic elastase was evaluated by in-cubating the AMCF with each enzyme at a final concentra-tion of 10
gg/mlfor 30 min at 37°C. The papain, trypsin,
andchymotrypsin wereobtained from Sigma Chemical Corp., the elastase and collagenase were obtained from
Worthing-ton Biochemical Corp.,Freehold, N. J. After thisincubation, theenzymes were inactivated by heating for 15min at 100°C.
The pH stability of the AMCF was evaluatedbyadjusting
the pH of the supemates containing the AMCF to 1.0, 3.5, 7.4, 9.0, and 12.0 with 1 N HCI or NaOH. After 1 h of
incubation, the pH of the supemates wasreadjusted to 7.4. The solubilityofAMCFinorganic solventswasevaluated byextracting 5 ml of the macrophage supemates containing
AMCFtwice with equal volumes of ethyl acetate, ether, or hexane. Extraction into ethyl acetate was tested with the
macrophage supernates adjusted to pH 3.5, 7.4, or 12.0; the
ether andhexaneextractions were performed with the macro-phagesupernate at pH 7.4. After eachextraction, theorganic
solvents were dried with a stream of N2 and the extracted
materialresuspendedin 5 ml of RPMI-1640. Thismaterial,as
well as the remaining aqueous phages, were then tested for chemotaxis for neutrophils as described above. Before testing,N2wasbubbled throughthe aqueousphasesto remove any residual organic solvent; the
with ethyl acetate were readjusted to pH 7.4. In all cases,
>90% of the AMCF activity in the original macrophage supernates was recovered in the organic plus aqueous phases.
To determine whether supernates of the stimulated macrophages contained hydroxyl-derivatives of 5,8,10,14-eicosatetraenoic acid(HETE) (24),AMCF present in 100 ml
ofa supernate of 108 stimulated alveolar macrophages was extracted into ethyl acetate and evaporated to a volume of
200 j.l with a stream of N2 as described above. The
super-nates were then analyzed for the presence of HETE by gas
chromatography-mass spectrophotometry (GC-MS) in the samemannerasdescribedbyHambergand Samuelsson (25). Briefly, the extractwasfirsttreatedwith an excess ofethereal diazomethane and then, after evaporation of the solvents, the residue was silylated using an excess (200 ul) of bistri-methylsilyltrifluoroacetamide. A 2-,ulaliquotof thissolution
(1% of the total volume) was subjected to GC-MS analysis
using a 3% OV-17 column programmed at 1°C/min, with an LKB-9000 combined GC-MSspectrometer.
Theeffectof aserum-derived chemotacticfactorinhibitor
on AMCF was tested by preparing the chemotactic factor inhibitor from normal human serum as describedby Beren-berg and Ward (26). The chemotactic factor inhibitor (0.1) was mixed with macrophage supernate (0.4 ml), incubated
at 23°C for 30 min, and thentestedforchemotactic activity
in thenucleopore filterassay.
neutrophils to follow a path of directed migration, the
ability ofAMCF to induceneutrophilstorelease a variety of
products was evaluated. Three neutrophil products were evaluated (27, 28); lysozyme, 8-glucuronidase, and
lacto-ferrin. For comparison, the release of lactic acid
dehydro-genase(LDH),acytoplasmic marker,wasalso determined. To evaluate the influence ofAMCF on these neutrophil products,1-5 x 107neutrophils(900 ul in HBSS) were placed in 12 x 75-mm plastic tubes containinga3-pm chemotactic filter in the bottom of the tube (29). Supernates (100
from macrophages stimulated with Sepharose-4Bwere then added(yieldingafinaldilution of themacrophagesupemate
of 1:10), and the cellswereincubatedat37°C for 0, 30, 60,or 120min.Supernatesof theneutrophil cultureswerethen
har-vested by passing the culture contents through 0.45-.um filters to remove the cells. As controls, supernates from unstimulated macrophages wereincubated withthe
neutro-phils as was media alone. The supernates from the
stimu-lated macrophageswereevaluatedfor lysozyme,,f-glucuroni-dase,lactoferrin, andLDH.
Lysozyme activitywas determined by measuringthe rate
ofchange ofturbidity ofa solution ofMicrococcus
lysodeik-ticus (Worthington Biochemical Corp.) at pH 6.2 (30).
,f-Glucuronidasewas assayed by measuring the release of
phenolphthalein from /8-glucuronate (Sigma Chemical Co.) after 6 h of incubation at pH 4.5 (31). Lactoferrin was
quantitated by radial immunodiffusioninagarosegels using rabbit antisera to human lactoferrin and purified human colostral lactoferrin (Calbiochem-Behring Corp., American
Hoechst Corp., San Diego, Calif.) as a standard (32). LDH
activitywasdeterminedbymeasuringtherateof decrease of absorbanceat 340 mmresulting from the oxidation ofNADH
duringconversionofpyruvate tolactate (33).
All statistical comparisons made using the two-tailedt test.
Ability of microorganisms and noninfectious
par-ticulates to stimulate alveolar macrophages to releaseAMCF. Supernates
of normal human alveolar
culture for3 h did not contain
cultured with heat-killedmicroorganisms
for normal human
of each of the particles with
significantly increased their abilityto
release of the chemotactic factor from
alveolar macrophages (Fig.1; P <0.01,
allcompari-sons). Opsonization of the particles notonly increased
of chemotactic factor released by
also increased therate
re-lease of the factor from thecells
This increased chemotacticactivity was
by the macrophages, becausenone
particles alone (culturedwithout
Unstimulated macrophagesmaintained in cul-ture
for0-4 h didnot
producethe chemotactic factor (Fig. 2). In comparison,
MACROPHAGE MACROPHAGE MACROPHAGE MACROPHAGE
ALONE + + +
S. ALBUS ZYMOZAN SEPHAROSE -4B
FIGURE1 Neutrophil chemotactic activityin supernates of control alveolar macrophages (O) compared with macrophages
stimulated invitro for 3 h by opsonized(U)or nonopsonized
(X)S. albus, zymosan, or Sepharose-4B. Neutrophil
chemo-taxis isexpressed as the number of cells per high power field
(hpf) reaching the lower surface ofa micropore filter. The data represent the mean+SEM of three separate experiments.
opsonized S. albus generated significant amounts of thechemotactic factor after2hofculture, with maximal production being reached in 3 h. Interestingly, after 18 h
of culture,supernates from the unstimulated
2 3 4
DURATION OF CULTURE (hours)
FIGuRE 2 Neutrophil chemotactic activity in supernates of control alveolarmacrophages(0- -0)ormacrophages stimu-latedfor variousperiodsoftimein vitroby opsonizedS. albus
phages contained levels of chemotactic activity
com-parable to that of supernates derived from stimulated
macrophages. The production of chemotactic factor by
control macrophages cultured for 18 h can likely be
ascribed to the stimulating effect of attachment and
spreading on the culture dish; unstimulated
macro-phages kept in suspension for 18 h (by tumbling in
plastic tubes) produced no chemotactic factor.
Macro-phage adherence to a plate was not critical for
of the chemotactic factor, because stimulated
macrophages kept in suspension by tumbling in
tubes produced AMCF (data not shown).
The ability of particulates to stimulate the
macro-phages to produce the chemotactic factor seemed to be
associated with either phagocytosis of the particulateor
attachment of the macrophagesto
For example, the macrophages phagocytized S. albus
and zymosan and they attached to the Sepharose-4B, all
of which stimulated the macrophages to produce the
phagocytized nor attached to untreated ORBC
supernates ofthe macrophages plusORBC
chemotactic activity (Fig. 3).
Ability of immune
release the chemotactic
release the chemotactic
generate quantities of
op-MACAPHAGE MACROPHAGE MACROPHAGE MACROPHAGE MACROPEAGE MACROPHAGE
ALONE +* + + +
E 19SEA 19S EAC 7S EA BSA-anti BSA
FIGuRE3 Neutrophil chemotactic activity in supernates of
control alveolar macrophages compared with macrophages incubated in vitro for 3 h with ORBC (E), ORBC coated with IgM (19S EA),SRBCcoated with IgM andcomplement (19SEAC),ORBCcoatedwith IgG(7S EA),orsoluble
num-ber of cellsperhighpowerfield(hpf) reachingthe lower
sur-face ofamicropore filter. The data represent the mean+SEM
ofthree separate experiments.
sonized zymosan particles (Fig. 3). The ability ofim-mune
be specific forIgG;
IgM-coated ORBC did not generate the
chemotactic factor. This specificity for IgG was
ability of the IgG-immune
with the macrophagesas
untreated ORBC nor IgM-coated ORBC formed
the macrophages. In contrast, theIgG-coated ORBC formed rosettes
phago-cytized by, the macrophages. However, the stimulationof
the macrophages by the IgG-ORBC complexeswas
due to the
fact that the IgG-ORBC
com-plexes were capable of interacting with the
macro-phages, since SRBC coated with IgM and complement
the macrophages but were not ingestedand did
chemotactic factor release.In
macro-phages and they were effective as IgG-coated ORBC in
of the chemotactic factor (data
of the stimulated macrophages fulfilled the
beinga chemotactic factor
When the supernatewas
present on both sides of the
without a gradient), it increased the random
proportionto its con-centration
However, when thesuper-nate was
only on the stimulus side of the
migrating through the filter at each concentration
level were greater than that due to randommigra-tion
of the relative activity of thechemo-tactic
neutrophils, monocytes, and
evaluate the relative chemotactic activity
of the chemotactic factorwere
the micropore filter. The chemotactic factor
chemotactic activity for
monocytes (Fig. 4).To
perspective, the chemotactic activity of6
of the activity of the endotoxin-activated humanserum
of the activity ofthe
preferential chemotactic activity ofAMCF
Capacityof theAMCFtoDeactivateNeutrophils andMononuclear CellstoSubsequent
prein-cubated withvarious Media
amountsof AMCF alone* AMCF FMLPt AS§
+ 0 1AMCF' 281 198 215 205
+6,u AMCF 20 56 49 66
+30I,AMCF 21 31 42 48
+O ,ju AMCF' 15 68 55 180 +6 ul AMCF 18 56 52 166 +30IL AMCF 12 52 49 124
j +-t -| *RPMI-1640 (150
T + + tN-formyl-Met-Leu-Phe (50nM; 150jzl).
5 Endotoxin-activated humanserum(5%, 150 AIl).
"&Neutrophilsand mononuclear cells were incubated with
0 1.5 6 30 150
varying amountsof theAMCFbeforetheir subsequent stimu-AMOUNTOF CHEMOTACTIC FACTOR (1I) lation with various chemotactic factors.
Neutrophilsormononuclear cellsreachingthelower surface
the AMCF. Variousamounts of the chemotactic factor
(gen-eratedby macrophages exposedtoSepharose-4B)weremade
to atotal volumeof 150 pi andplaced inthe lower well of
thechemotaxischamber,andperipheralbloodmononuclear cells (80% lymphocytes-20% monocytes) (O 0) or
neutrophils( -0)wereplacedintheupperchamber. The
resultsareexpressed asthe number ofcellsperhighpower
field (hpf) reaching the lower surface ofamicropore filter.
To compare the
relativeactivity of the chemotactic
eosinophils,PMN cell sus-pensions containingboth of these cell typeswere
peripheralblood ofthree individuals with
used. Both methods of
analysisdemonstrated that the chemotactic factor
Partial characterization ofthe chemotactic
factor.Greater than 80% of the chemotactic activity of the
macrophagesupernates was stable to
56°Cfor30 min and
100°Cfor30 min,at pH 1.0, 3.5, 9.0, and 12.0for60 min, andafterexposure to papain (10
min, 37°C), collagenase(10
4g/ml,30min,370C), and elastase (10
mac-rophages (which didnotcontain
the chemotactic factor)
each ofthe enzymes
de-scribed above. However,
chemo-of the nucleopore filters in responsetovarious chemotactic factors.
tactic factor with a
inhibitorresultedin 90%decrease inthe chemotactic
Characteristics of theHumanAMCF
Percentageof Conditions originalactivity*
Chemotactic factor 100
+ Heating (30 min,56°C) 105
+ Heating (30 min,100°C) 95
+pH 1.0(60 min) 98
+pH 3.5(60 min) 105
+pH 9.0(60 min) 97
+pH 12.0(60 min) 99
+Chemotactic factor inhibitor t 10
+Trypsin (10,ug/ml,30min,37°C)5 94
+Collagenase (10 ug/ml,30min,37°C)5 98
Media alone 0
*The percentage of original activitywasestimatedby
deter-miningthe dilution of the chemotactic factor which resulted
ina50% maximal migration of neutrophils inthe chemotaxis assay.
§ After incubation with thechemotactic factor. Theenzyme
wasinactivatedby heatingat100'C for 15min.
-100 200 300 400 500 600 700 25 50 75 100 125 150
ELUTION VOLUME (ml)
FIGURE 5 Molecularweightestimationof the human AMCF. The chemotacticfactorwas
gen-erated by alveolar macrophages exposed to Sepharose4B particles. (A) Sephadex G-25 column chromatography; (B) Bio-Gel P-2 column chromatography. Both columns wereelutedat
4°C with 40mM PBS, pH 7.4. Neutrophil chemotaxis is expressed asthe number of cells per
high power field (hpf) reaching the lower surface of the micropore filter. Molecular weight markers include: blue dextran (2 x 106),bacitracin (1,450), vitaminB12(1,350), and[3H]thymidine
fractionation of the
chemotactic factor revealed that themajor
(>75%)of the chemotactic activity of AMCF eluted withanapparentmolecular
weight of400-600 (Fig. 5).
Isoelectric focusingof the AMCF (400-600
peaks ofactivity at pl, 7.6
lipid.Extraction into organicsolvents revealed that 75% of AMCF activity was removed from the
pH 3.5, 80% with
ethylacetate at pH
pH12, 60% with etheratpH
7.4,and 45% with hexane at pH 7.4
(Table III).The activity that was removed from the
was recovered from the respective organic solvents.
chemotacticfactor present in supernates of
of culture exhibitedsimilar solvent partition characteristics to the chemo-tacticfactor whichwaspresent in supernates of
shown).Because atleastaportion of AMCF
were further evaluated for the presence of
hydroxyl-derivatives of HETE
pathwayof arachidonic acid which are known to be chemotactic for
ng/mllevel. Since a 1:10 dilution of the
neutrophilsataconcentrationof less than 1
ofAMCF in our
FIGURE 6 Isoelectric focusing of the AMCFin ahorizontal
bed ofgranulated gel withapH gradient of 2.5-9. The pH
afinalconcentrationof 2%(wt/wt)inthe gel slurry.AMCF, partially purified by prior gelfiltration,wasaddedtothe
granu-lated gel bed(3% vol/vol) beforeapplication of the electro-focusingcurrent(25 W, 5°C,16h).Thegelwasthencollected
as75-mmstripsproceeding from anodetocathode and each gel fractionwaselutedwith 3 mlof0.85%NaCI-40mM
phos-phate, pH 7.4. The eluate from the gel strips was then
assayedforneutrophilchemotacticactivity.Gel fractions from control experiments that did not contain AMCF demon-strated that the granulatedgel-ampholytefocusing bed itself didnotcontainmeasurableneutrophilchemotactic activity.
Extraction of the AMCFintoOrganic Solvents
Percentage of original activity Solvent Condition extracted*
Extractedintoethylacetate pH3.5 75
pH 7.4 80 pH 12.0 25
Extracted intoether pH 7.4 60
Extracted intohexane pH 7.4 45
by determining the dilution of the chemotactic factor that resulted in a 50% maximal migration of neutrophils in the chemotaxis assay.
ifHETE is present, it is at a concentration 2.5 x 10-'
than theminimum 1
,ug/ml level suggested by
Effect of the macrophage-derived chemotactic
macro-phage chemotactic factor also stimulated human
7). Additional studies
the lysozyme and lactoferrinfound in
cultures of macrophagesupernates
derived from the
of the supernates
demonstrated that: (a)
lysozymewere present, it
of that foundin
supernates plus neutrophils; and (b)no
the accumulation of
that the alveolar
modulating neutrophilmigration to
ofAMCF canbe triggered by microorganisms,
noninfectiousparticulates, and immune complexes, each
of whichare models
for lungdisorders asso-ciated with
neutrophilaccumulation in the lower res-piratory tract (5-14). Because this
of these pathogenicagents, its
forthe accumulation of neutrophils in
chemotactic factoris not
spontaneously from unstimulatedalveolar
macro-phagesis consistentwith the observation that
rarelyfound in the
lungsof normal in-dividuals.
linesof evidence demonstrated that the macrophage-derived chemotactic factor was not related to any
(34-37).First, it could be
producedin the absence of serum or any serumcomponent. Second, it was not inactivated by an anti-C5a
antibody, demonstratingthat it was not
shown) (37). Third,the small molecular
weightof the chemotactic factor
serum-derived chemotactic factorsthat are generated as aresult of activation of the complement system, Hage-man factor, or the
(34-37).These observations also suggest that AMCF was not derived from the action of
macrophage-producedpro-teases on serum components in a manner analogous to PMN protease digestion of IgG (38) or
themacrophage-derived chemotactic factor was related to the presence
NEUTROPHILS NEUTROPHILS CONTROL MACROPHAGE MACROPHAGE SUPERNATE
SUPERNATES CONTAINING CHEMOTACTIC
FIGuRE7 Release of lysozyme (O), f-glucuronidase *, LDH (0), and lactoferrin (0J) from neutrophils exposed for 2h to supernates from unstimulatedmacrophages("control") and supernates frommacrophagesstimulated with Sepharose-4B. The supernates from the macrophages stimulated with Sepharose-4B contained the chemotacticfactor, whereas the supernates from the unstimulatedmacrophages did not. As-says for the release oflysozyme,f-glucuronidase,and LDH were performed with 107 neutrophils; "100% release," that amount of lysozyme, 13-glucuronidase, and LDH pres-ent in107neutrophils.The assays for the release of lactoferrin were performed with 5x 107 neutrophils; "100% release,"
chemotactic products of bacteria (16, 17), because
cultures of the lavage fluid from all of the normal
volunteers were sterile as
were the macrophage
Partial characterization of AMCF suggested thatit
small (400-600 mol wt) and
distinct peaks of
chemotactic activity were obtained
following isoelectric focusing (pl 7.5, 5.2).In
the partial solubility of theAMCF in
solvents suggests thatat
activity may be derived
Although AMCF appears to
be at least partially lipidin
nature, it is
from HETE (a previously
de-scribed lipid chemotactic factor for neutrophils) (24).
supernates of stimulated
macro-phages demonstrated that, ifHETE was
these supernates, it was at a concentration so low that
it could not possibly have
accounted for the neutrophil
chemotactic activity ofAMCF.
demonstrate that the majority of the chemotactic
from alveolar macrophages,at
Valone et al. (40), utilizing techniques
different from those usedto
the present study, demonstrated that rabbit alveolar
capable of producingHETE
The activity of the AMCFwas
both acid and
alkaline pH and
could be inactivated
in-hibitor described by
Berenberg and Ward (26),
physiologic mechanisms mayexist in vivo to
regulate macrophage induction of
chemotactic factor derived from human alveolar
closely resembles the small molecular
weight neutrophil chemotactic factors derived from
(3) and guinea pigs (4),
if any, species differencesexist. It is
known, however, whether theseAMCF are
dis-tinguishable from the small molecular weightchemo-tactic
factor preparations (41).
influenced the migration of several types of
mononuclear cells. However,its
exceeded that of
mononuclearcells. This is in contrast to the
of the small molecular
cells (ECF-A), which
of theAMCF was not ECF-A. The low molecular
that elute from Dowex-1 columns (Dow Coming
pH3.2 to 2.3
of AMCF demonstrated no
chemo-tactic activity at these isoelectric points. A low
molecular weight (300-400) chemotactic factorwith
activity for eosinophilic
has also been isolated from extractsto
bronchogeniccarcinomas and a renal cell carcinoma
metastatic to the
lung (44). This factor differed from
ECF-A derived from mast cells in that it elutedfrom
at pH 5.5 to 4.5 rather than 3.2 to 2.2.
focusing of the AMCF revealed a similar
chemotactic activity with a pI of 5.2; however,
the partial solubility of theAMCF in
demonstrates that at least a portion of thechemotac-tic
the factors derived from lung tumors. In addition,
it is clear that the
cell of origin of all of the
chemo-tactic activity presentin
differs from that of the
the lung tumors. Finally, the more
basic chemotactic molecules (pI 7.6) that are present in
different from the ECF derived
both random migration as
directed migration (chemotaxis) of neutrophils,
fulfilling the acceptedcriteria
for being a
factor. However, the macrophage supernates not
stimu-lated neutrophils to release lysozyme and lactoferrin.
of interest, becauseit
stimu-lated alveolar macrophage not only can amplify the
by recruiting blood leukocytes
by stimulating those leukocytes to dischargeat
least some of their stored products.
for generation and
release of the
wide variety of
capable of triggering the release of the
factor from alveolar macrophages. Each of
these stimuli hasa common
with the macrophage membrane.In
albus, zymosan particles,
Sepharose-4B, and extended attachmentto a
plastic surface, thenature
of thisinteraction is not
factor releaseseems to
through the IgG-Fc receptor, because IgMEAC
receptor. Interestingly, however,C3b on
particulates does appearto increase the release
Sepharose-4Ball cause chemotactic
beyondthat due to the
pro-duce the chemotactic factor, it is importantthat these stimuliare representativeofa spectrumof pathogenic agentsthatcauseneutrophil accumulationinthe lower respiratory tract.Thus, the macrophage seems tohave multiple means by which it can interact with patho-genicagents:first,itcanphagocytizethe agent;second,
itcan destroy itby secretingavarietyofenzymes and other toxic materials; and third, it can enlist the aid of polymorphonuclear leukocytes by secreting a low molecular weight chemotactic factor.
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