Effects of bafilomycin A1 on functional capabilities of LPS ... - CiteSeerX

Effects

of bafilomycin

LPS-activated Akhil

A1 on functional

alveolar

Bidani

Departments

Internal

A. Heming

Medicine

and

Physiology

&

Biophysics,

University

of

Abstract: Resident alveolar macrophages (m) possess plasmalemmal vacuolar-type W-ATPase (V-ATPase) that plays a crucial role in regulation of intracellular pH (pH1). To assess the importance of this V-ATPase to m4 effector functions, resident alveolar m from rabbits were activated with E. coli-derived lipopolysaccharide (LPS) and exposed to bafilomycin A, a specific inhibitor of VATPase. Bafilomycin caused a significant cytosolic acidification in both the absence and presence of C02HC03, and in both unstimulated and activated m. Su-

m with the oxa-i,3-diazole pH to fall by of H extrusion

peroxide production and F receptor-mediated phagocytosis also were reduced in bafilomycin-treated m. Similar effects were elicited by acidifying the cytoplasm in the absence of bafilomycin, by lowering extracellular pH (pH0) from 7.4 to 6.5-6.6. Thus, the effects of bafilomycm on phagocytosis and superoxide production probably were related to cytosolic acidification, secondary to blockade of V-ATPase-mediated W extrusion across the plasma membrane. Conversely, bafilomycin significantly increased TNF-a release. This effect cannot be explained by a bafilomycin-induced acidosis because acidic pH0 significantly reduced TNF-a release. The results demonstrate that V-ATPase activity is an important determinant of the effector functions of LPS-activated m4. J. Leukoc. Biol. 57: 275-281; 1995.

ester-activated

Key .

Words: tumor

intracellular

necrosis

factor

pH

phagocytosis

superoxide

#{149}

vacuolar-type

.

anion

H-pump

alveolar

that

functions

The

functional phagocytic

ity,

nals,

macnophage to

and

(m)

maintain

prowess capability,

production

is a mononuclear

the

sterility

of

of phagocytes recognition

and

release

of

mediators

pH

intracellular radicals also

by

by

of

release

supetoxide

vacuolar-type

alveolar ATPase

responsible

principle

mechanism

[9,

to tumor [7,

H-ATPase

the the

12].

Under

being

of m,

in

pH homeostasis dependent on

m in mediates

exclusively

m of

production

Intracellular m is markedly

chemotaxis

penitoneal

inhibitors

would of

hypothesis, Murphy Cl reduces superoxide zymosan-activated documented a similar

extracellulan necrosis 8]. the

(V-ATPase)

foi for

these

setting pH1

of

supenoxide

effects

production,

A1

from

on

TNF-a

the

pH

of

bafilomycin

inhibitor of Vlipopolysaccharide from rabbits were incubated with E. and LPS-activated receptor-mediated

and

release

sensitivity

MATERIALS

AND

Preparation

of alveolar

TNF-a

release,

in

was

opposite

of this

to

functional

that

attribute

METHODS

Resident alveolar m4 iavage of New Zealand as described previously washed

twice

H, and

cellular

with

macrophages were white [9,

a

obtained by bnonchoalveoiar rabbits (body mass 2.0-2.5 12]. The recovered cells

nominally

Vis

is

BCECF,

Abbreviations:

I

resident

from

the

the absence and presence of bafilomycin A1 (10 jzM). Bafilomycin A1 caused a significant decrement in steadystate pH1, reduced phagocytosis and superoxide production, and increased TNF-a release. The effects of bafilomycin A1 on phagocytosis and superoxide production were in the same direction as the pH sensitivity ofthese functions and, hence, probably were related to the cytosolic acidification that resulted from blockade of V-ATPase-mediated H extrusion across the m plasma membrane. In contrast, the effect of

me#{128}liati eflective

serum;

In

treatment

this

m. investigated

A1, a macrolide antibiotic and specific ATPases [13], on select functions of (LPS)-activated alveolar m4. Resident m obtained by bronchoalveolan lavage and coli-denived LPS for 18-24 h. Unstimulated m were assessed for steady-state pH, F

acids inhibits a (TNF-a),

C02-HC03, produced pH,

pH-

with

Fonman [6] have shown that NBDproduction in phonboi esterand alveolar m. Swallow et al. [5] effect with bafilomycin A in phorbol

studies

phagocytosis,

the

Consistent

C02-free

kg), were

physiological

salt

cx-

and penitoneal of plasmalemmal 9-11].

to influence

m.

and

penitoneal present

EC50.

[6,

recovery

is

m4

Similarly,

baseline

conditions,

by

anion

alveolar

factor

alveolar activity

nominal absence effiux of metabolically

are the by

inhibited

[3-6].

be predicted

alveolar

cytotoxic

superoxide and

pH

in

and

mobilsig-

of these attributes pH. For example, are directly affected

Production

decrements

pulmonary

phagocytosis,

loads

with

[2].

neutnophils,

inhibited

posune and

(pHi),

acidification

Texas

lining.

pivots on their of extraceliular

agents (reviewed in nell 1). Several known to be sensitive to acidic chemotactic responses of neutrophils cytosolic

alveolar

Galveston,

V-ATPase inhibitors, 7-chloro-4-nitrobenz-2(NBD-Cl) [6] or bafilomycin A1 [12], causes 0.3-0.5 units, presumably due to blockade across the plasma membrane. It follows that

functions

The

Branch,

and, therefore, cannot be explained by the bafilomycininduced decrement in pHi. Overall, the data emphasize the importance of V-ATPase activity for m effector functions.

phagocyte

the

Medical

sensitive

predicted

INTRODUCTION

Texas

V-ATPase

bafilomycin

The

of

macrophages

and Thomas

of

capabilities

the acid

HEI1ES,

2 ,7 -biscarboxyethyl-5,6-carboxyfluorescein;

E,,

close;

,

membrane

potential;

FBS,

fetal

N-[2-hydroxyethylpiperazine-N’-[2-ethanesulfonic

bovine acidi;

I..PS, lipopolysaccharide; in, tuacrophage: N 111)-Cl, 7-chloro-4-nitrobenz-2-oxa-l,3-diazole: pH, intracellular pH: pH.,, extracellular pH; P55, physiological salt solutiotl: TNF-a, tumor tiiecliaii

necrosis

inhibitory

factor

Reprint ternal

a:

concent

V-ATPase,

requests: N’Ie(licine,

rat ion;

Akhil

vacuolar-type Biclani,

University

of

H’-ATPase.

Pulmonary iixas

I)ivision, Medical

Branch,

I)epartmnent

of

In-

Galveston,

TX

1995

275

77535-0561.

of alveolar

R-crised

Journal

July

of Leukocyte

13,

1994:

tCCel)ted

Biology

September

Volume

8,

57,

1994.

February

solution CaCl2,

(P55) containing (in 1 MgSO4, 2 K-phosphate,

mM)

135 NaCl, S D-glucose,

[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic

pH

=

in

The

7.4).

cells medium

a complete

S KC1, 1 and 6 N-

acid]

then

(HEPES;

were resuspended ( 106 (RPMI-1640) containing

cells/ml) either 25

mM

NaHCO3 (referred to as HCO3-RPMI) on 25 mM (referred to as HEPES-RPMI; pH = 7.4 in both cases). The resulting suspensions contained >95% viable cells (trypan blue exclusion) of which >96% were m4 ( Papanicoiaou and Giemsa staining and positive staining for nonspecific estenase activity) [9]. Some of the m (termed freshly-isolated) were studied within 2-4 h of their isolation. The remaining cells were plated in sterile tissue culture

HEPES

2.7 KC1, 8.1 were scraped cells. three

flasks and incubated for 18-24 h at 37#{176}Cin an humidified atmosphere containing 0 or 5% CO2. The incubated cells were divided into two treatments. One treatment group (termed LPS-activated) was exposed to LPS (serotype 0SS:BS from Escherichia co/i); see below for details of LPS concentration and duration of exposure. The second treatment group (termed unstimuiated) was not exposed to LPS. Unstimulated and LPS-activated m were recovered by scraping the incubation plates with a rubber policeman to remove adherent cells and centrifuging the media. The viability of unstimulated and LPS-activated m4 was >95%.

Determination

of baseline

ratio

(peak/isosbestic)

was

calibrated

to

standard K-nigenicin technique [14]. To determine the relationship between lular pH (pH0), BCECF-loaded cells were mm

in

RPMI-1640

with

uned at wavelength

pH1

7.4).

using

pH0

of

6.6

pH1 and extracelpreincubated for on

7.4

(37#{176}C, 0

nary

A

same

-=

2 mm.

recorded

conditions.

were held (95% air) suspensions

Bafilomycin for

an

The

resulting

cell

suspen-

M)

period

was

added

of

and

pH1

of cellular

was

10 mm.

ATP concentration

LPS-activated m (18 h, 10 g/ml) were mm in HEPES-RPMI with pH0 of 6.5 extracts then were prepared for ATP analysis, as described previously [15]. The [ATP] was measured with a luminometen (Monolight 2010, Analytical Luminescence Laboratory, San Diego, CA) using a lucifeninluciferase assay described previously [15].

Phagocytosis using

276

of F receptor-mediated was

quantified

fluorescein-labeled

Journal

of Leukocyte

under

E.

of superoxide

colz

Biology

with

CO2-fnee

Volume

conditions

to

the

57,

>90%

protocol

February

washed a Coulter

were then lysed with of the lysate meas500

nm

and model

emission

F-2000,

anion

anion

production

radicals was reduction

measured as the of cytochnome c

then

were

incubated

for

1 h

in

sterile

15-mi

studies

demonstrated

that

F0F1-type production

by

for tumor

NaN3

alone,

H-ATPase,

had

LPS-activated

necrosis

which

no

alveolar

factor

inhibits

effect

on

su-

m.

a

serum

(FBS),

(pH0 plates

100 7.4).

=

U/ml The

and incubated and nonadherent The adherent m4

HCO3-RPMI

on

HEPES-RPMI

penicillin,

and

100

tg/ml

plated in sterile for 2 h (37#{176}C, 5% C02). cells then were removed were washed and covered

m4

were

(pH,,

=

6.5

or

7.4)

supplemented with 100 U/mi penicillin, 100 g/ml streptomycin, 0 or 100 ng/ml LPS, and 0 or 10 tM bafilomycin. The m4 then were incubated for 24 h in an humidified atmosphere containing 0 or 5% CO2, as appropriate (37#{176}C). The supernatants (i.e., m-conditioned media) were collected and stored at -20#{176}C for subsequent assays of TNF-a. Preliminary studies indicated that m viability remained

phagocytosis similar

m

streptomycin multiple-well The supernatent and discarded.

Freshly-isolated and preincubated for 30 or 7.4 (37#{176}C). Cell

Measurement

wavelength of (spectrofluonometer

ofsuperoxide dismutase-inhibitable

The

Bioassay

bovine

Measurement

by centnifugation, and counted using

FL). The m4 the fluorescence

nm

tubes then adherent

standard cytotoxicity assay with a TNF-sensitive L929-fibroblast cell line (ATCC CCL 1; American Type Cultune Collection, Rockville, MD) was used to assess the titer ofTNF-a released by alveolar m4 in the presence on absence of CO2-HCO3 [18]. The m were suspended in HCO3-RPMI (106 cells/mi) supplemented with 10% fetal

in room air on a flowing stream during measurements of pH. was recorded for a period of

A1 (10

additional

excitation of 525

mitochondnial

on

in

the

an

Measurement

peroxide

under

II (Hialeah, X-100 and

assay remove

Hitachi Instruments Inc.). In order to correct for adherent but non-internalized bacteria, the fluorescence intensity values obtained with cells at 1#{176}C were subtracted from the paired values at 37#{176}C.The resulting phagocytic index was expressed as arbitrary fluorescence units/104 m/30 mm.

C02). As appropriate, the m then were transferred to CO2-free P55 on to a CO2-containing PSS (composition in mM: 115 NaCI, 25 NaHCO3, S KC1, 1 CaC12, 1 MgC12, 2 Na-phosphate, and 5 D-glucose; equilibrated with 5% CO2 air)

m were recovered in saline (as above),

Multisizen 10% Triton

The to

7.4).

=

policeman

centrifuge tubes in a shaking water bath (37#{176}C). The supernatants were collected by centnifugation and analyzed spectrophotometnically for reduced cytochnome c (absorbance at 550 nm; Vanian DM5 300, Walnut Creek, CA). Supenoxide production was calculated using an absorption coefficient for reduced cytochnome c of 21 mM cm [17]. NaN3 was included in the incubation medium in an attempt to inhibit possible neoxidation of the reduced cytochnome c. Prelimi-

5%

sions (106 cells/mI) of 5% CO2 gas The pH of cell

The times

pH

under CO2-fnee conditions [17]. Freshly-isolated, unstimulated, and LPS-activated (18 h, 10 tg/ml) m were suspended in CO2-free PSS (106 cells/mi) supplemented with 2 mM NaN3, 80 M cytochnome c, 0 or 60 g/m1 superoxide dismutase, and 0 on 10 tM bafilomycin A1 (pH0 = 6.5 or

the

30

K-phosphate; with a rubber

Production supenoxide

pH

Intracellular pH was measured using the pH-sensitive fluorescent probe, 2 ‘ ,7’ -biscanboxyethyi-S,6-canboxyfluonescein (BCECF). Unstimulated and LPS-activated m (18 h, 10 tg/ml) were loaded with the probe by incubation with the acetoxymethyi ester form of BCECF (S M in RPMI-1640) for 30 mm. BCECF fluorescence intensity was monitored at 37#{176}Cusing excitation wavelengths of 504 nm (peak fluonescence) and 430-435 nm (isosbestic point), and an emission wavelength of 527-530 nm (spectrofluorometers; model F-2000, Hitachi Instruments Inc., San Jose, CA and model RF-S000, Shimadzu Corp., Kyoto, Japan). The fluorescence intensity

described by Oben and Foreman [16]. The E. co/i were opsonized with rabbit polyclonal IgG, washed twice, and resuspended in HEPES-RPMI. Freshly-isolated, unstimulated, and LPS-activated (18 h, 10 tg/ml) m were incubated with opsonized bacteria (final ratio m:bactenia = 1:50) and bafilomycin A1 (0-10 jIM) for 30 mm in sterile 15-mI centnifuge tubes in shaking water baths at 1 or 37#{176}C(pH0 = 6.5 on 7.4). Phagocytosis was terminated by addition of 10 volumes of ice-cold saline (composition in mM: 137 NaC1,

1995

during

the

assay.

Data

The cytotoxic bioassay was a modification of that described by Flick and Giffond [18]. Briefly, confluent monolayens of L929 cells were established by incubating cell suspensions

(4

x

10

cells/mi

in

Mi99

medium

sup-

analyses data un(F)

RESULTS Figure

1A

shows

steady-state

the

pH1

of

relationship

between

unstimulated

and

pH,

and

LPS-activated

the

m

in

the presence of 5% CO2. At pH0 7.4, the pH of unstimuiated cells averaged 7.16 ± 0.04 (n = 8). Macnophage pH decreased significantly with decrements in pH,, in the presence ofS% CO2. These data are consistent with the published pH-pH() relationship of alveolar m4 under C02-free conditions [12]. The pH of LPS-activated cells tended to be more acidic than that of paired unstimulated cells, but the

actinomycin D alone and an internal standard consisting of recombinant munine TNF-a (specific activity = 40 units/pg). Mi m-conditioned media were assayed in duplicate. TNF-a units were defined as the reciprocal of the diiution of m-conditioned media that was required to produce a 50% decrement in absonbance, relative to the blank. It should be noted that m-conditioned media contained bafilomycin A1 in addition to released TNF-a. With the dilution sequence used in the present studies, the L929 cells were exposed to 3.3 M bafilomycin A1.

and

and

The data are presented as arithmetic means ± SE. The were statistically analyzed using one-way paired and paired Student’s t tests, as appropriate. A probability value of 0.05 was used to evaluate statistical significance.

plemented with 2% FBS, 100 U/mi penicillin, and 100 g/ml streptomycin; pH,, = 7.4) in sterile multiple-well plates for 24 h (37#{176}C, 5% CO2). The supernatants then were discarded and the monolayers were covered with M199 medium supplemented with 100 U/mI penicillin, 100 tg/ml streptomycin, and S sg/ml actinomycin D (pH0 = 7.4). Aliquots of m4-conditioned media were added to each well in serial dilution. The L929 cells were incubated for a second 24-h period (37#{176}C, 5% CO2) and then fixed and stained with 5% crystal violet. Dye uptake was determined with an ELISA reader at 590 nm. Each assay included a blank consisting of

Chemicals

presentation

effects were Bafilomycin

not

acidification

statistically A1 (10 tM)

in

and

in both

1B).

Similar

both

the for

sence

of

CO2

stimulated

and

of

presence

bafilomycin

than

in

at

pH,,

cells

A1 rabbit

[12]. acidosis its 7.4,

a

significant

and

unstimulated

sence of CO2-HCO3 bafilomycin-induced

reagents

unstimulated

absence

effects

previously

significant. caused

presence.

cells

of C02-HC03

(Fig.

have

alveolar

The absolute tended to For

bafilomycin

cytosoiic

LPS-activated been

described

m

in

magnitude be larger in example, A1

(10

the

with riM)

ab-

of the the abun-

caused

an sence presence cells at ment

0.48-unit decrement in steady-state pH1 in the abof CO2 but only an 0.24-unit decrement in the of CO2 (P < 0.05). Similarly, with LPS-activated pH,, 7.4, bafilomycin A1 caused an 0.27-unit decrein steady-state pH in the absence of CO2 and an 0.17-unit decrement in the presence ofCO2. Figure 2A illustrates the time course ofthe bafilomycin effect under C02free conditions (pH0 = 7.4). The bafilomycin-induced acido-

BCECF and fluorescein-labeled E. coli were purchased from Molecular Probes (Eugene, OR). HEPES was purchased from Research Onganics (Cleveiand, OH). RPMI-1640 was purchased from GIBCO (Grand Island, NY). Actinomycin D was a kind gift of Merck, Sharpe & Dohme (West Point, PA). Recombinant munine TNF-a was purchased from Genzyme (Cambridge, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO).

*

.c U) C

:

A

+

a-

7.2

_J

B

U)

0.6

C

I I

5% CO2

C02-free

pH0

7.4

pH0

6.5

*

7.0 *

0.4

6.8 E

U) C

6.6

:i:

to

0.

U)

a-J

+

0.2 6.4

0.0

6.2

pH0

6.6

pH0

7.4

Fig. 1. Effects of bafilomycin A, (Baf. 10 sM, 10-mm treatment) on the steady-state pH of alveolar m. Cells were preincubated for 18 h in the presence (LPS) or absence (Unstim) of 10 sg/ml LPS at pH, 7.4 and then for 30 mm at pH,, 6.6 or 7.4. A) steady-state pH, in the presence of 5% CO2 (mean values ± SE: n = 4-8). *Significantly different from paired value for unstimulated cells (P < 0.05). ‘Significantly different from paired value for m at pH,, 7.4 (P < 0.05). Intracellular pH was not significantly affected by LPS (P > 0.05). B) absolute magnitude of the bafilomycin-induced decrement in pH, (pH) under a variety of conditions. Data points are mean values ± SE (n = 3-4). ‘Significantly different from zero (P < 0.05). Bafilomycin A, caused a significant acidosis in all cases.

Bidani

and

Hemint

Bafliomycin

effects

on

macrophage

functions

277

Baf

A

B

7.4

0.6

7.2

0.4 I

I

7.0

0.

0.

0.2 6.8

0.0

6.6 0

150

300

450

Time Fig. HCO,

2. Kinetics (pH

ruin,

in(lependent

ilieflt

in

ihe

pH

and

dynamics

7.4).

,=

of the eflective

occurred

1.2

±

are

efl#{232}ctson

pH,,

concentration

=

7.4).

(EC50)

pH

m4

A) time

.

of experiments

of hafilomycin

C02-free,

A. Data

was

B) dose-response

points

0.7

with are

mean

course

concentration

(150)

V-ATPases 150 values

is cannot

bafulomycin-induced

influenced compensatory well as by The

the rates capabilities the affinity

cellular

[ATP]

and

0.10

1.13

A1

for

membrane-

membrane to the

acidosis

protein [13]. EC50 for the

because

the

latter

is

of metabolic acid production and the ofother acid-base transporters, as of the V-ATPase for bafilomycin A1. ofalveolan

IATPI The

±

bafliomycin

nmol/mg be compared

celiular

by

activation. 1.42

of

0.4

±

m in

was

unchanged

freshly-isolated

by cells

0.18 nmol/106 cells 4). In LPS-activated

LPS was

at pH0 7.4 and cells, the [ATP]

6.5, respectively (n = was 1.26 ± 0.06 and 1.37 ± 0.03 nmol/106 cells at pH0 7.4 and 6.5, respectively (n = 3). We have shown previously that 10 iM bafilomycin A1 has no effects on the [ATP] of alveolar m JCo-incubation of alveolar m with fluonescein-labeled E. co/i (pH,) = 7.4, nominal absence of CO2) yielded phagocytic indices of3.7 ± 0.5 and 3.7 ± 1.0 units/104 m/30 mm for freshly-isolated and unstimulated cells, respectively (n = 9). Prior activation of m with E. co/i-derived LPS caused a marginal, but not statistically significant, increase in phagocytosis (5.0 ± 1.1 units/104 m/30 mm for LPSactivated cells; n = 11). In all cases, phagocytosis was significantly A

(10

inhibited

30-mm

tM,

by

concurrent

treatment)

3).

1

of

the

bafilomycin-induced

A1]

acidification

in

the

10

100

absence

of COy

(.iM) nomiiial

from

0.8-1.7

deereA1).

To assess

bafilomycin A (10 tM, 60-mm treatment) significantly inhibited supenoxide production by 50-65% (Fig. 4). TNF-a release from unstimulated cells was 287 ± 63 (n = 13) and 417 ± 139 (n = 4) units/ml/24 h in the presence and absence of CO2, respectively (pH,, = 7.4 in both cases). The release of TNF-a increased 2- to 4-fold following LPS activation (1092 ± 99 and 1065 ± 271 units/ml/24 h with LPS-activated cells at pH,, 7.4 in the presence and absence of CO2, respectively; n = 4-19). Extracellular acidification (pH0 6.5, 5% CO2) reduced the release of TNF-a by LPS-activated cells by 53 ± 5% (n = 6) (Fig. 5). TNF-a release was significantly increased by concurrent exposure to bafilomycin A1 (10 M, 24-h treatment) in all cases, except for unstimulated cells in CO2-fnee medium at pH,, 7.4 (Fig. 5).

I

100

1

7.4

pH0

pH0

6.5

*

80

C,)

T

U)

0 >,

60

C)

0 0) Co

.c 40

0.

to bafilomycin the pH sen-

exposure

(Fig.

0.1

.sM.

0.1

bound These

0.01

3 different animals. The half-time of the response ranged from relationship. pH is the absolute magnitude of the bafilomycin-induced values ± SE (n = 3. except for single experiment with 100 sM hafilomycin cells

relatively rapidly; the average half-time was mm (n = 10 using 0.1-100 tM bafilomycin A1). The median effective dose (EC50) for the cellular acidosis was 0.7 tM bafilomycin A1 (Fig. 2B). The reported median inhibitory

0.001

[bafilomycin

representative

concentration

(nominally

Iiie(lian

siN

traces

0

750

(sec)

of i)afilomycin

The

600

>

of phagocytosis, LPS-activated cells also were studied at pH,, of6.S in the absence ofbafilomycin A1. Extracellular acidification inhibited phagocytosis by 24 ± 5%, relative to paired values at pH0 7.4 (Fig. 3). Superoxide anion production by freshly-isolated cells averaged 4.4 ± 1.2 nmol/l06 cells/h (n = 5; pH,, = 7.4, nominal absence of C02). Supenoxide production increased to sitivity

9.5 ± 2.2 nmol/106 m/h (n without LPS and to 17.6 ± 2.1 18-h incubation with LPS (10 cases). Superoxide production significantly

Further,

278

reduced

at

Journal

both

when

pH0

6.5

of Leukocyte

pH0

and

4)

=

after

18-h

nmol/106 m/h jg/ml) (pH0 by LPS-activated was

7.4,

Biology

lowered

concurrent

Volume

(n

=

7.4

m to 6.5 (Fig. exposure

57,

February

0 Fresh

Fig.

3.

( open ( 30-mm

8) after in

20

a:

both

was 4). to

1995

(Unstim),

bar; relative ±

1.1

different from

control)

treatment,

pressed (5.0

Unstim

F, receptor-niediated

stimulated

incubation

=

CU

C)

to

from paired

LPS-activated

and

presence = 6.5 or

paire(1

values

at

control pH,

7.4

freshly-isolated alveolar

(solid bar) 7.4, nominally control




munine

TNF-a

bafilomycin

A1.

(100-1000

units/mi)

Bafilomycin

A1

together

with

increased

the

0-10

M

apparent

titer

of recombinant TNF-a by 5.3 ± 0.7% in the presence M of the inhibitor (n = 4) and by 10.2 ± 2.2% presence of 10 M bafilomycin A1 (n = 4). During the unements

of

3.3

tM

m

TNF-a,

the

bafilomycin

A1.

L929

Thus,

cells the

were

effects

A i on L929 cells were an order of magnitude plain the observed increase in TNF-a release treated LPS-activated m4.

in

200

C) Co

*

of

1 150

the

meas-

exposed

of

.

to

bafilomycin

iEIL

100

.

too small to cxby bafilomycin-

CU

50

a: 0

Unstim

DISCUSSION Fig.

Alveolar supenoxide

m

to E. co/i-derived and release

responded production

responses

are

characteristic

of

m

by

caused

phonbol

Swallow

and

hibits

V-ATPase

nine)

by

nitric

m

a transient

ester

co-workers

oxide.

the they

to

that

respiratory

have

that

found

that

effect

in-

m

pH0

penitoneal

Consistent

with

pH0

7.4

this

6.5

80

-I-

0

observation, of resident

(NBD-Cl

of intact A1 causes

60

I

0.

is

C)

40

m