ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2002, p. 4013–4015 Vol. 46, No. 12
0066-4804/02/$04.00⫹0 DOI: 10.1128/AAC.46.12.4013–4015.2002
Copy igh © 2002, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Up ake and In acellula Ac i i y o Linezolid in Human
Phagocy es and Nonphagocy ic Cells
A
´l a o Pascual,* So ía Balles a, Isabel Ga cía, and E elio J. Pe ea
Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Se ille, Spain
Recei ed 13 No embe 2001/Re u ned o modi ica ion 19 June 2002/Accep ed 12 Augus 2002
The in acellula pene a ion and ac i i y o linezolid in human polymo phonuclea leukocy es and issue-
cul u ed cells (McCoy) we e e alua ed. Linezolid eached in acellula concen a ions sligh ly g ea e han
ex acellula ones in bo h ypes o cell. The up ake was apid and no sa u able and was a ec ed by en i on-
men al empe a u e and cell iabili y. Linezolid showed sligh in acellula ac i i y agains S aphylococcus
epide midis a high ex acellula concen a ions.
The pene a ion and in acellula ac i i y o an imic obial
agen s in phagocy es a e pa icula ly impo an unde condi-
ions in which he in ec ing mic oo ganism is able o su i e
and mul iply. Se e al in ec ing bac e ia, howe e , a e able o
mul iply wi hin nonphagocy ic cells, pa icula ly epi helial
cells. Fo hese in ec ions, he use o agen s capable o accu-
mula ing and emaining ac i e in such cells could be o po en-
ial clinical in e es (9). The high in acellula accumula ion o
an an imic obial agen does no always mean good in acellula
ac i i y. I is impo an , hen, o e alua e whe he in acellula
an imic obial agen s emain ac i e agains bac e ia. Linezolid
is an o al oxazolidinone an ibac e ial agen ha ac s by inhib-
i ing bac e ial p o ein syn hesis (4, 13–15) and has a wide
spec um o ac i i y agains g am-posi i e o ganisms includ-
ing me hicillin- esis an S aphylococcus au eus, penicillin- and
cephalospo in- esis an pneumococci, and ancomycin- esis-
an En e ococcus aecalis and En e ococcus aecium, as well as
anae obes such as Clos idium spp., Pep os ep ococcus spp.,
and P e o ella spp. (1, 5–7). Linezolid is bac e ios a ic agains
mos suscep ible o ganisms, bu displays bac e icidal ac i i y
agains some s ains o pneumococci, Bac e oides agilis, and
Clos idium pe ingens. (2, 3). The e is no in o ma ion abou
he in acellula pha macology o hese compounds in human
phagocy es and nonphagocy ic cells. The pu pose o his s udy
is o e alua e he up ake o linezolid by phagocy es (polymo -
phonuclea leukocy es [PMNs]) and nonphagocy ic cells (Mc-
Coy). The in acellula ac i i ies o his compound agains
S aphylococcus au eus and S aphylococcus epide midis we e
also e alua ed.
The up ake o adiolabeled linezolid (167 ␮Ci/mg; Pha ma-
cia.) by phagocy es and nonphagocy ic cells was de e mined by
a eloci y g adien cen i uga ion echnique, as desc ibed by
Klempne and S y (8). In hese expe imen s, phagocy es o
issue-cul u ed cells we e incuba ed in Hanks’ balanced sal
solu ion con aining di e en concen a ions o linezolid (1 o
40 ␮g/ml). A e di e en pe iods o incuba ion a 37°C, he
cells we e sepa a ed om he ex acellula solu ion by cen i -
uga ion h ough a wa e -impe meable silicone oil ba ie in a
mic ocen i uge ube. A 10-␮l aliquo o he ex acellula me-
dium and he en i e cell pelle , ob ained by cu ing o he
po ion o he mic ocen i uge ube con aining he pelle , we e
placed in 3 ml o scin illa ion luid (Ready Mic o; Beckman
Ins umen s, Inc., Fulle on, Cali .) and coun ed wi h a liquid
scin illa ion coun e (model LS 1801; Beckman). The in acel-
lula wa e space was measu ed by using i ia ed wa e and he
ex acellula ma ke [
14
C]polye hylene glycol (1.4 mCi/g; Am-
e sham In e na ional, Plc., Buckinghamshi e, Uni ed King-
dom). The cells we e incuba ed wi h hese adiolabeled com-
pounds o 2 min a 37°C, and hen he cells we e sepa a ed
om ex acellula luid by eloci y g adien cen i uga ion and
coun ed wi h a liquid scin illa ion coun e . The o al wa e
con en o he cell pelle was co ec ed o apped ex acellu-
la wa e (i.e., polye hylene glycol space) o ob ain he in a-
cellula wa e space. The accumula ion a e o he an imic o-
bial agen in PMN (cell-associa ed d ug) o issue-cul u ed
cells was calcula ed and exp essed as a cellula /ex acellula
concen a ion (C/E) a io (11). The da a a e exp essed as
means ⫾s anda d de ia ions. Di e ences be ween g oups
we e compa ed by a iance analysis, used o assess s a is ical
signi icance a Pⱕ0.05.
Figu e 1 shows he kine ics o he up ake o linezolid by
human PMNs and McCoy cells. The in acellula pene a ion
o linezolid was apid, eaching in acellula concen a ions 1.2
imes highe han he ex acellula concen a ions a e 20 min
o incuba ion in bo h kinds o cells. A e 180 min o incuba-
ion, he C/E a io alues emained he same o McCoy cells,
bu had dec eased by a ound 50% in PMNs. To e alua e
whe he linezolid ha had been aken up by human PMNs was
igh ly bound o cellula componen s, we e alua ed he kine ics
o e lux (Fig. 2). The elu ion o linezolid om human PMN
was apid. A e 10 min o incuba ion in an an imic obial- ee
medium, 90% o linezolid was eleased. The in acellula pen-
e a ion o linezolid in o PMN and McCoy cells was no sa u-
able a ex acellula concen a ions anging om 1 o 40 mg/
li e . The C/E a ios in PMNs anged om 0.9 (ex acellula
concen a ion, 40 mg/li e ) o 1.3 (ex acellula concen a ion,
20 mg/li e ). A he same ex acellula concen a ion, he C/E
a ios anged om 1.1 (ex acellula concen a ion, 40 mg/
li e ) o 1.3 (ex acellula concen a ions, 1, 5, and 20 mg/li e )
in McCoy cells.
* Co esponding au ho . Mailing add ess: Depa amen o Mic obi-
ology, School o Medicine, Uni e si y o Se ille, A da. Sa´nchez Piz-
jua´n s/n, 41009-Se ille, Spain. Phone: 34.5.55008287. Fax: 34.5.4377413.
E-mail: [email p o ec ed].
4013
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Fu he s udies o elucida e he mechanisms o linezolid
up ake by PMNs we e pe o med as desc ibed p e iously (10).
The in luence o en i onmen al empe a u e (4 e sus 37°C),
cell iabili y, and pH (5 o 8) and he e ec s o human se um,
pooled se um (5 and 10%), and me abolic inhibi o s (1.5 ⫻
10
⫺3
M [each] sodium luo ide and sodium cyanide; Sigma
Chemical Co., S . Louis, Mo.) on he up ake o linezolid by
PMNs we e e alua ed. The in acellula pene a ion o lin-
ezolid in PMNs was also measu ed a e s imula ing he cells
wi h 200 nM pho bol my is a e ace a e (PMA; Sigma) and
a e phagocy osis o opsonized s aphylococci (in 5% pooled
human se um a a 10/1 a io o bac e ia o PMNs) and opso-
nized zymosan (0.9 mg/li e ; Sigma) (Table 1). Cell-associa ed
linezolid dec eased in iable cells a 4°C (C/E a io, 0.75 ⫾
0.15; con ol, 1.2 ⫾0.4) and inc eased signi ican ly (P⬍0.05)
when dead cells a 37°C we e used (C/E a io, 1.98 ⫾0.15). A
possible explana ion o his inc ease migh be ha he o ma-
lin used o kill PMNs caused s uc u al changes in he PMNs,
which a o ed nonspeci ic binding o he linezolid. This hy-
po hesis is cu en ly unde in es iga ion. The up ake o his
new oxazolidinone was no a ec ed by ex e nal pH (which
anged om 5 o 8). Concen a ions o pooled human se um
and di e en me abolic inhibi o s (NaCN and NaF) did no
a ec he in acellula pene a ion o his an imic obial agen .
Nei he s imula ion o he PMNs by a memb ane ac i a o
(PMA) no phagocy osis o opsonized S. au eus and zymosan
signi ican ly a ec ed he in acellula pene a ion o linezolid.
Mos da a indica ed ha a possible passi e mechanism was
in ol ed in he in acellula pene a ion o his agen (7, 12).
To e alua e he in acellula ac i i ies o an imic obial
agen s, a p e iously desc ibed me hod was used (11). S. au eus
ATCC 25923 and S. epide midis ATCC 41134 we e used o
killing assays. Suscep ibili y s udies we e de e mined by mi-
c odilu ion assay. The MICs o linezolid o hese s ains we e
2 and 1 mg/li e , espec i ely. The da a we e exp essed as
pe cen ages o su i ing s aphylococci compa ed wi h he con-
ol le els (wi hou an imic obial agen s) a 3 h. In addi ion o
de e mining bac e ial su i al, mo phological s udies we e also
ou inely pe o med, a ime ze o and a e 3ho incuba ion,
o e alua e he disposi ion o bac e ia (cell associa ed o ex-
acellula ). All assays we e pe o med in duplica e wi h PMNs
om i e di e en dono s. The da a a e exp essed as means ⫾
FIG. 1. Kine ics o linezolid up ake by human PMNs and McCoy cells (n⫽4). Expe imen s we e ca ied ou a ex acellula concen a ions
o 10 mg/li e a 37°C. Da a a e exp essed as means ⫾s anda d de ia ions.
FIG. 2. E lux o linezolid om human PMNs (n⫽3). A e incu-
ba ion wi h 10 mg o linezolid pe li e o 20 min, he cells we e
washed and esuspended in an imic obial agen - ee medium. Cell-
associa ed linezolid was hen measu ed a di e en imes.
TABLE 1. E ec s o a ious condi ions on in acellula
pene a ion o linezolid in human PMNs
a
Condi ion C/E a io
b
Con ol ( iable cells, 37°C, pH 72) ......................................1.20 ⫾0.40
Viable cells, 4°C......................................................................0.75 ⫾0.17
c
Dead cells, 37°C......................................................................1.98 ⫾0.15
c
pH
5 ............................................................................................1.30 ⫾0.42
6 ............................................................................................1.13 ⫾0.171
7 ............................................................................................1.18 ⫾0.39
8 ............................................................................................1.20 ⫾0.14
Sodium cyanide.......................................................................1.25 ⫾0.21
Sodium luo ide ......................................................................1.20 ⫾0.14
Human se um
5%.........................................................................................1.37 ⫾0.12
10%.......................................................................................1.47 ⫾0.14
Opsonized PMA .....................................................................1.25 ⫾0.21
Opsonized S. au eus ...............................................................1.35 ⫾0.13
Zymosan................................................................................... 1.3 ⫾0.27
a
The condi ions examined include en i onmen al empe a u e, cell iabili y,
ex e nal pH, me abolic inhibi o s, se um, memb ane ac i a ion, and phagocy osis
o S. au eus and zymosan.
b
Expe imen s (n⫽4) we e ca ied ou o 20 min a ex acellula concen a-
ions o 10 mg/li e .
c
P⬍0.05 compa ed wi h he con ol.
4014 NOTES ANTIMICROB.AGENTS CHEMOTHER.
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s anda d de ia ions. Di e ences be ween g oups we e com-
pa ed by a iance analysis, which was used o assess s a is ical
signi icance a Pⱕ0.05. A ex acellula concen a ions a ain-
able in human se um, linezolid did no a ec he in acellula
su i al o s aphylococci. A high ex acellula concen a ions
(10 and 20 mg/li e ), linezolid showed sligh in acellula ac-
i i y agains S. epide midis, bu no agains S. au eus (Fig. 3).
The di e ences in he in acellula ac i i y migh be ela ed o
di e ences in he in insic ac i i y o his compound agains
bo h s ains.
In summa y, linezolid pene a es bo h phagocy es and non-
phagocy ic cells, eaching in acellula concen a ions sligh ly
g ea e han he ex acellula ones. Linezolid showed sligh
in acellula ac i i y agains S. epide midis a high in acellula
concen a ions.
This wo k was pa ially suppo ed by a g an om Pha macia.
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FIG. 3. Ac i i y o linezolid agains in acellula S. au eus and S.
epide midis in human PMNs (n⫽4). Da a a e exp essed as pe cen -
ages o su i ing bac e ia a e 3ho incuba ion compa ed o hose o
he con ols wi hou linezolid (means ⫾s anda d de ia ions). *, P⬍
0.05 compa ed wi h con ol.
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