Mechanisms involved in Helicobacter pylori—Induced inflammation

GASTROENTEROLOGY

1993:105:1431-1440

Mechanisms Involved in Hekobacter pylori-Induced Inflammation NORIMASA YOSHIDA,* D. NEIL GRANGER,* DOYLE J. EVANS JR.,* DOLORES G. EVANS,* DAVID Y. GRAHAM,* DONALD C. ANDERSON,§ ROBERT E. WOLF,* and PETER R. KVIETYS* *Departments of Physiology and Medicine, Center of Excellence for Arthritis and Rheumatology, Louisiana State University Medical Center, Shreveport, Louisiana; *Department of Medicine, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas; and “Upjohn Laboratories, Kalamazoo, Michigan

Back&ound: Helicobacter pylori infection is associated with mucosai inflammation. The aims of the present study were to assess whether a water extract of H. pylori promotes neutrophil (polymorphonuclear leukocyte [PMN]) adherence to endotheliai cells and define the molecular basis of this adhesive interaction. Methods: lntravitai microscopy was used to study leukocyte adhesive interactions in rat mesenteric venules in situ. PMN-endothelial ceil adhesive interactions were studied in vitro using human PMNs and monolayers of human umbilical vein endotheiiai ceils (HUVEC). Results: In vivo, superfusion of rat mesentery with the H. pyloriextract increased leukocyte adhesion and emigration in venules. In vitro, adhesion of human PMNs to HUVECwas increased by the H. pylori extract in a concentration-dependent manner. Pretreatment of HUVEC atone with H. py/ori extract had no effect on PMNadherence, whereas pretreatment of PMNalone significantly increased their adherence to HUVEC.The extract-induced adhesion was significantly diminished by monoclonal antibodies (MAb) directed against either CD1 la, CD1 1 b, or CD18 on neutrophiis, and by MAbs against intercellular adhesion molecule-l (ICAM-l), but not Eor P-selectin, on endotheliai cells. Conclusions: These studies suggest that products of H. pylori elicit gastrointestinal infiammation by promoting PMN adhesion to endothelial ceils via CDlla/CD18and CDllb/ CD18-dependent interactions with ICAM- 1.

S

everal

lines of evidence

in the pathogenesis

ation.

There

implicate

of gastric

is a very

strong

Helicobacterpylon

and duodenal association

ulcer-

of active

chronic gastritis and duodenitis, as well as gastric and Ingestion duodenal ulcers, with H. pyiori infection.‘-’ of the bacteria by human infection and histologically Eradication of the results in resolution tion is associated ease.3,4,7,8 The exact contributes to the

volunteers produces gastric demonstrable gastritis.“*”

organism in patients with ulcers of the pathology, whereas reinfecwith greater recurrence of dismechanisms by which H. pyioti pathogenesis of gastroduodenal

ulcers

is unclear

but may include

degrade

and injure

mucin’*

reducing

the resistance

Alternatively, and activate

chemotactic

and other

of the latter injury

of neutrophil ies have possess

are directly

shown

is the observation

cells, including present

material

promotes

leukocyte

venules.

human

adhesion

Using

neutrophils

to identify

which

an in vitro two

active

role in neutrophil-endothelial by the H. pyloti extract.

Neutrophil by

adhesion

various

CD 1 l/CD1

coordinately

mediator

of neutrophil

adhesion

a variety of in vivo and in vitro tion. The CD1 l/CD18 complex

of

played

an

cell interactions cells

regulated

complex

has been shown

in mesen-

consisting

cell types

l&and-receptor inflammatory

8 glycoprotein

neutrophils

of H. pylon

cells, we were able

to endothelial

complexes that initiate response to appropriate vated

system

and endothelial of these

In the

the cell surface)

and emigration

elicited tated

inflammatory

and monocytes.‘8-21 from

stud-

of H. pyLori

for various

released

of the

the extent recent

study, we show that a water extract

(containing teric

with

or sonicates

activity

neutrophils

and severity

Furthermore,

that extracts

chemotactic

cells,

tissue injury.‘,*,5

correlated

‘M

that attract

inflammatory

causing

hypothesis

infiltration.

to acid injury.

that H. pylori pro-

that the degree of H. pylori infection mucosal

that

ce1ls,‘3T’4 thereby

substances

leukocytes

and indi-

substances

of the mucosa

neutrophils

the activated

In support

epithelial

it has been suggested

duces and releases with

both direct

H. pylon’may produce

rect components.

is faciliadherence

interactions in stimuli.22-25 The expressed

by acti-

to be an important to endothelial

cells in

models of inflammaconsists of three het-

Abbreviations used in this paper: FITC, fluorescein isothiocyanate; fMl.P, N-formyl-methionyl-leucyl-phenylalanlne; HESS, Hanks’ balanced salt solution; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-l; LPS, lipopolysaccharide; LTB,, leukotrlene B,; MAb, monoclonal antlbody; PAF, platelet-actlvating factor; PMN, polymorphonuclear leukocytes. 0 1993 by the American Gastroenterologlcal Association 0016-5085/93/$3.00

1432

YOSHIDA ET AL.

erodimers, logically

each of which distinct

P,-subunit unique

(CD18)

At least

the most important

three

adhesion

glycoproteins

cells have been implicated

inflammation.22

Intercellular

1) is constitutively

cell surface

ligand for both

CDlla/CD18

and CDllb/CD18.

is not basally expressed

on quiescent

teracts with specific X)

on

neutrophils

ICAMimal

to

and E-selectin

surface

expression

tion.22,24 Endothelial tory

stimuli

P-selectin, rides present

initiate require after

which

adhesion.23-25

endothelial

cell

to promote

adhesion.23

In the

approach

was

of each of the

determinants

to the neutrophil-endothelial

mobilizing

with oligosaccha-

contribution

molecular

activa-

to inflamma-

minutes)

study, an immunoneutralization

aforementioned

Both

several hours for max-

(within

the relative

it in-

(e.g., sialyl Lewis

also can interact

on neutrophils

used to define

where

cells also respond

by rapidly

endothe-

by cytokines,

to the cell surface

oligosaccharides

E-

of adhesion

cell interactions

elicited

by the H. pylori extract.

Surgical (200-250

procedure.

and fasted for 18 hours before experiments. were initially anesthetized with thiobarbital body wt), and then a tracheotomy

The animals (12 mg/lOO g

was performed to facili-

tate breathing during the experiment.

The right carotid ar-

tery was cannulated, and systemic arterial pressure was measured

using

a Statham

transducer connected line abdominal mid-jejunum

(Oxnard,

CA)

incision

pressure

was made, and a segment of the

was exteriorized

through the abdominal

sion. All exposed tissue was moistened gauze to minimize evaporation

lntravital

P23A

to the carotid artery cannula. A mid-

microscopy.

inci-

with saline-soaked

and tissue dehydration. Rats were placed in a supine

position on an adjustable Plexiglas (Rohm and Haas, Philadelphia, PA) microscope pared for microscopic

stage, and the mesentery was pre-

observation as described previously.”

The mesentery was draped over an optically clear overslip that allowed for observation

of a 2-cm* segment of tissue.

The exposed bowel wall was covered (Dow Chemical Co., Indianapolis,

with Saran Wrap

IN), then the mesentery saline (pH

H. pyfoti-induced

gastritis. The or-

mimicked that measured in situ.28 An inverted microscope observe the mesenteric

scribed.26

(VK-C150;

consisted

of

Bacto

(Diaphot-TMD;

Nikon, Japan)

with a X40 objective lens and a X10 eyepiece was used to

ganism was grown on blood agar plates as previously demedium

rats

that the p0, of the fluid bathing the mesentery (40 mm Hg)

which was isolated from a gastric antral biopsy of a volun-

growth

Sprague-Dawley

on a purified laboratory diet

7.4) that was bubbled with a mixture of 5% O,, 5% CO,, and

pylon extracts were prepared from strain 8826,

The

Male

g) were maintained

90% N,. The superfusion rate was set at 1 mL/min to ensure

H. pylori Water Extracts

teer with asymptomatic

In Vivo Studies

was suffused with warmed bicarbonate-buffered

Materials and Methods H.

of the

ner.

in

on

properties

ginosa, and Helicobacterfeliswere prepared in an identical man-

adhesion

expressed

and filtration

water extract. Extracts from Escberikhia coli, Psetldomonasaera-

adhe-

and appears to serve as the

lial cells, but when the cells are activated is mobilized

het-

which consists

indicated the centrifugation

steps had no effect on the proadhesive

cell in-

cell

material,

mainly of membrane vesicles and whole flagellae. Preliminary experiments

and the CDllb/

of neutrophil-endothelial

1 (ICAM-

E-selectin

to their

CD1 lb/CD18,

I

the endothelial selectin

according

in neutrophil-endothelial

on endothelial

molecule-

weight complex

considered

involved

during

filter. This procedure removes much of the

high molecular

CDlla/CD18

the modulation sion

syringe-adapted

an d are classified

CD 18 are generally teractions.22,24

of an immuno-

(CD1 1) and a common

i.e., CD1 la/CD18,

and CDllc/CD18. erodimers

is comprised

a-subunit

a-subunit,

expressed

GASTROENTEROLOGY Vol. 105, No. 5

Hitachi,

microcirculation.

Japan)

mounted

A video camera on the microscope

brain-heart infusion, 0.5% Bacto (Difcor, Detroit, MI) yeast

projected the image onto a color monitor (PVM-2030;

extract, 2.0% Bacto agar, and 7% fresh horse blood. Plates

Japan), and the images were recorded using a video cassette

were inoculated, and the bacteria (passage 1-12) was grown

recorder (AG-1960;

in a CO, incubator, with 12% CO, and 100% humidity, for 48 hours. Cells were harvested with sterile cotton swabs into

Single unbranched venules with diameters of 25-35 pm and a length of >150 pm were selected for study. Venular

distilled water, using 1.0 mL per plate (109-10’o

diameter (D”) was measured using a video caliper (Microcir-

bacteria).

Sony,

Panasonic, Japan).

for 20

culation Research Institute, Texas A&M University, College

at 12,000 rpm (17,OOOg) for

Station, TX). The number of adherent leukocytes was deter-

15 minutes. The resultant supernatant is the initial water extract; no preservatives were added, and the extract was stored at -20°C until needed. Before use, the water extract

mined off line during playback of videotaped images. A leukocyte was considered adherent to venular endothelium if it remained stationary for a period of 230 seconds. Ad-

was brought to room temperature and centrifuged at high speed (18,000 rpm [38.7 k X g] for 20 minutes), and the pellet discarded. The supernatant was then passed through a 0.2 micron Acrodisc (Gelman Sciences, Ann Arbor, MI)

herent cells were expressed as the number per 100 ym length of venule. The number of emigrated leukocytes was also determined off line during playback of videotaped images. Leukocyte emigration was expressed as the number

The cell suspension was kept at room temperature minutes before centrifugation

H. PYLORI-INDUCED

November 1993

per microscopic

field (I. 7 X lo-’ mm2 or 150+m

length

of

and

98% pure

(acetic

acid-crystal

1433

violet

stain-

ing).

venule). Centerline

red blood

cell velocity

(V,,)

velocimeter

(Microcirculation

using an optical

Doppler

search Institute,

Texas A&M University)

rotating

glass disk

blood blood

exclusion)

NEUTROPHIL ADHESION

coated

with

flow was calculated cell velocity

microvascular ometry.

the product

= centerline

(V,,,,

definition,

Experimental sured on line (arterial

Venular

provided

red

have been previously

described.

and

immunoglobulin

velocity/l.6)”

After

pressure,

were recorded

ge-

determinant

based on

framework

MAb W6/32

cylindrical

all parameters velocity,

mediately

thereafter,

pylon’ extract buffered

on videotape

the mesentery

(final dilution

venular

was superfused

saline (PBS) for 40 minutes,

and repeat measurements final 10 minutes

with H.

in phosphate-

with video recordings

of all parameters

of perfusion

Im-

made during

the

pended

2 X 10’ cells/ml phi1 suspension

Subjects.

twice

dothelial

cells and neutrophils

tional

Review

State

University

written

Board

Medical

consent

cells (HUVEC) lagenase

cells.

Human

were harvested

treatment

as previously 199 (GIBCO,

Inc.,

Sigma Chemical

Logan,

with

heparin

(100 IU/mL

penicillin,

and 0.125 pg amphotericin factor (80 pg/mL,

MA). The cell cultures fied atmosphere taining Primary

with

0.02% through (0.1%)

tissue culture

and

passage

25 pg/mL

labelling

by brief trypsaline con-

were

[EDTA]). seeded

into

1 l-mm, as endothe-

at confluence

(Biomedical

and

is 95%-98%

counter.

to the HU-

(cpm)

(cpm)

X 100

+ Wash

(cpm)

+ Lysate

(cpm)

In some experiments, monolayers. washed

viable

After

the extracts

a 30-minute

to remove

the

extracts,

added to the monolayers

monolayers,

neutrophils

cules

washed,

on neutrophils

assays. The MAbs and the extract)

to HUVEC

later.

lmmunofiuorescence H. py/orz’ extract

were pretreated

with the HUVEC

or endothelial

30 minutes

were assessed.

added

directed

were added

(20 l.tg/mL)3’

adhesion to naive

by the H. pylon’ extract

MAbs

centrations

were

for the neutrophil-endothe-

elicited

by including

HUVEC neutrophils

assessed.

determinants

lial cell interactions identified

naive

and neutrophil

and adhesion

The molecular

were added to HUVEC

incubation,

on surface

to adhesion cells

(along

in the adhesion

with

monolayers

the neutrophils

at saturating

and neutrophil

adhesion

flow cytometry. expression

were mole-

con-

assessed

Effects

of the

of CD1 l/CD18

on

Technolo-

neutrophils was determined as previously described.3’ Briefly, the reaction mixture consisted of lo6 neutrophils, H.

factor VIII

pylon’ extract,

CA).

that

Lysate Supernatant

and used in adhesion

Neutrophils. Human polymorphonuclear leukocytes were isolated from venous blood of healthy adults

population

in a gamma

as follows3’:

experiments,

using standard dextran sedimentation and gradient separation on Histopaque 1077 (Sigma).3’*32 This procedure yields a PMN

and the

of the supernatant,

was quantitated

for 30 minutes,

Inc., Stoughten,

MA) and mouse antihuman

La Jolla,

H.

the super-

were washed,

that adhered

In other

cell growth

The cells were identified

with Dil-Ac-LDL,

gies Inc., Stoughton,

monolayers

at a neutro-

(30 minutes),

assessed

in

Labeled

or without

neutrophils

extracts

streptomycin,

fibronectin-coated

appearance

of added

(230 mg/L;

acid

HUVEC

monolayers

the monolayers

and lysate were

were to re-

(HBSS).

of 1O:l with

cells lysed. The 5’Cr activity

fluid,

cells

and then resuspended

coincubation

was removed,

sus-

neutro-

The

salt solution

cell ratio

Sigma), anti-

buffered

plates (GIBCO)

confluent.

mg/L;

at 37’C in a humidi-

in phosphate

lial cells by their cobblestone

(PMN)

100 &/rnL

5% CO, and expanded

third

48-well

(Calbiochem,

(2.4

(10 IU/mL;

ethylenediaminetetraacetic

gelatin

positive

NY) supple-

thymidine

were incubated

(0.25% trypsin

assays when

The cells were

Island,

Technologies

Na5’CrOJmL

radioactivity

After

The percent

were

the cells at

cords by col-

B), and endothelial

Biomedical

in

provided

fetal calf serum (Hyclone

sodium

anti-HLA

cold PBS at 250 g for 8 minutes

in the study.

described.31

Utah),

MAb 4A5

the

neutrophils

30 l&i

were added to HUVEC

VEC

and

for 60 minutes.

neutrophils

wash

control

by incubating

Hanks’ balanced

vein endothelial

Co., St. Louis, MO), glutamine

JRH Biosciences),

sinization

subject

umbilical

with 10% heat-inactivated

Laboratories

Each

Grand

en-

at the Louisiana

from umbilical

in Medium

mented

biotics

Center.

human

by the Institu-

Research

and was paid for participating

Endothelial

plated

was approved

for Human

with

move unincorporated

natant used to obtain

Isolated

plasma-free

remaining

The procedures

MAb (mouse

the binding

(IgG2a)39 were used as controls

at 37°C

pylori extracts.

In Vitro Studies

P. McEver)38

A nonbinding

unknown),36

assays.

Phil-to-endothelial

with the extract.

(MAb)

by Dr. Rodger

in PBS and radiolabeled

washed

antibodies

assays.

Adhesion

mea-

for 10 minutes.

of X20) dissolved

Gl [IgGl]),

(neutrophil

were in a steady state, images from the mesenteric

preparation

ICAM-

the adhesion

erythrocyte

Monoclonal

CD1 lb (IB,), 33 CD1 la (TSl/22),% (R6.5),36 E-selectin (CL2),37 and P-se-

CD18

lectin (Gl; generously

r = S(V,,,JD,).”

protocols.

against

(LM2/1),35

of mean

area, assuming

prepared

against a

cells.

wall shear rate (2) was calculated

the Newtonian

diameter)

red blood

from

cross-sectional

Venular

calibrated

Re-

antibodies.

Mono&nal

was measured

(trypan

blue

and a MAb (murine

anti-human;

4 pg) against

either CD1 la, CD1 lb, or CD18 in a total volume of 500 PL HBSS. After a 30-minute incubation at 37°C neutrophils were

washed

with

PBS

and

fluorescein

isothiocyanate

(FITC) conjugated goat anti-mouse immunoglobulin was added at a final concentration of 8 pg/mL. After a 15-minute incubation at 4°C the neutrophils were washed with

1434

YOSHIDA ET AL.

GASTROENTEROLOGY Vol. 105, No. 5

PBS, and MAb binding to neutrophils was analyzed in a flow cytometer (EPICS 753; Coulter Electronics Inc., Hialeah, FL). Characterization of H. pylon’extract adhesive properties. To assess whether the inflammatory mediators, leukotriene B, (LTB,), platelet activating factor (PAF), or IV-formyl-methionyl-leucyl-phenylalanine (fMLP) could account for the proadhesive properties of the H. pylori extract, receptor antagonists were included in the adhesion assays. The receptor antagonists, SC 41930 (anti-LTB,; Searle, Skokie, IL), WEB 2086 (anti-PAF, Boehringer, Ingelheim, Germany), or iV-t-Boc-Met-Leu-Phe (anti-fMLP, Sigma) were used at a final concentration of 10e6 mol/L. Molecular sizing experiments were performed usingdialysis membranes (10,000, 15,000, 25,000, or 50,000 mol wt pore size; Spectrum, Houston, Texas). Two milliliters of the extract were dialyzed for 24 hours in 500 mL PBS at 4°C; the PBS was changed at 12 hours. A gel filtration approach was also used to estimate the molecular weight of the adherence factor(s) in the H. pyloti extract. Four milliliters of the extract was eluted through a column of agarose A- 1.5/m gel (Bio-Rad), collecting 2.5 mL fractions. Dialyzed extract or eluted fractions (agarose gel chromatography) were assessed for their proadhesive properties. To further characterize the adherence factor(s) contained in the H. py/oti extract, we performed the following maneuvers. The extract, or active fractions (agarose gel chromatography), were heated (56°C for 30 minutes or 1OO’C for 10 minutes) in a water bath. The extract, or active fractions, were exposed to two types of proteases: pronase (pronase E, type XIV, from Streptomycesggriseus; Sigma) or pepsin (pepsin A from porcine stomach mucosa; Sigma). The extract, or active fractions, were incubated with pronase (2 U/mL, final concentration) or pepsin (110 U/mL, final concentration) for 1 hour at 37°C. To assess whether the proadhesive activity of the extract, or active fractions, were acid labile, the pH of the material was reduced to 1.5 with HCI for 1 hour at 37°C. Statistical analysis. All values are expressed as means f SE. Data were analyzed using an analysis of variance and Student’s t test (with Bonferroni corrections for multiple comparisons).

Results Figure 1 summarizes the effects of H. pyioti extract on leukocyte adherence and emigration in rat mesenteric venules. Superfusion of the mesentery with the extract (20-fold dilution) resulted in an increased leukocyte adherence (threefold) and emigration (fourfold). These leukocyte-endothelial cell interactions could not be attributed to a decline in hydrodynamic dispersal forces,4o because venular wall shear rate (750.7 f 141.5 s-‘) was not signiIicantly altered by superfusion with the extract. When tested in the in vitro adhesion assay, the H.

HBSS

EXTRACT

HBSS

EXTRACT

10 -

5-

0-

Figure 1. Effects of superfusion of the rat mesentery with a 20-fold dilution ofthe H. pylori extract on leukocyte adherence (A) and emigration (f3) in 30-Frn venules. The mesentery was prepared for intravital microscopy as described in the text. The solutions were superfused for 40 minutes with video recordings made for playback analyses during the last IO minutes. Each value represents mean ? SE of 6-8 experiments. *P c 0.01 compared with HBSS.

pyloti extract

produced a concentration dependent increase in PMN adhesion to HUVEC (Figure 2). The proadhesive effects of the extract were apparent at a lOOO-fold dilution and produced maximal adhesion at a lo-fold dilution. The proadhesive activity of the extracts did not vary with increasing passage number

November

H. PYLORI-INDUCED

1993

H. pylon’extract

40 r

response

elicited

(5 to 6-fold

NEUTROPHIL ADHESION

a more

pronounced

1435

adhesion

than the H. fefis extract

increase)

(100% increase). Figure against

4 summarizes neutrophil

the effects

and endothelial

cules on the increased observed

in the presence

inhibited

by anti-CD18

effects of anti-CD1 x5

HBSS

x50

x10

Xl00

Xl000

ditive

x10000

of the extract

la and anti-CD1

when

rected against

endothelial

was completely

lb MAbs.

la and anti-CD1

elic-

adherence

and partially

used in combination.

mole-

to HUVEC

The augmented

MAb

directed

cell adhesion

PMN adhesion

ited by the H. pyLoti extract.

by anti-CD1

of MAbs

inhibited

The inhibitory

1 b MAbs were adThe only

cell adhesion

MAb

molecules

dithat

I

Helicobacter

Pylor~

Extract

(X dilution)

Figure 2. Concentration dependent effects of the H. oylori extract onPMN adherence to HUVEC. 5’Cr-labeled PMN were added to HUVEC monolayers, in the absence or presence of different dilutions of the H. pylori extract, and PMN adherence to HUVEC assessed 30 minutes later. Each value represents the mean f SE of 4-5 experiments in triplicate. *P < 0.001 compared with HBSS.

diminished

the PMN

anti-ICAM-

MAb;

were without

effect.

mouse

IgG,

(data

not

adherence

to HUVEC

was the

the anti E- and P-selectin Neither

the control

nor the binding,

shown)

showed

MAbs

nonbinding

nonfunctional

controls

any significant

inhibitory

effects. To assess whether

the hyperadhesion

induced

by the

H. pyLori extract (data

not

shown).

were prepared tested

Water

in an identical

in the adhesion

adhesion

extracts

responses

Figure

extracts

tended

to increase

VEC,

only the extracts

duced

statistically

3 compares

by extracts

E. coli, P. aeruginosa, and H. jks.

adherence

the

all of the to HU-

increases.

However,

were

individually washed,

posure

with

the extract

for 30

assays. Ex-

alone

to the extract

increased

to HUVEC

(Figure

5A). However,

exposure

of HUVEC (Figure

pretreated

and used in the adhesion

of neutrophils

adhesion

sion of naive

from H. fels and H. pyLori pro-

significant

cells,

minutes,

from H. py1or-i;

Although

PMN

bacteria

as H. pylon’and

fashion

assay.

elicited

of other

was due to activation of endothelial neutrophils, or both, either HUVEC or PMN

alone

to the extract

neutrophils

5B). Increasing

did not increase

to the treated the duration

adhe-

endothelium

of endothelial

cell

the 30 .

r

Untrueted

HESS

E.coli

P. oeru.

H.felia

CD18

CDlla

CDllb

CD;la

ICAM-

E-ml

P-ml

CDllb

H.pylori

I

I

NBP

Extracts Figure 3. Comparison of the proadhesive properties of water extracts of H. pylori,H.felis, P. aeruginosa, and E. co/i (all extracts were used at a IO-fold dilution). S’Cr-labelled PMN were added to HUVEC monolayers, in the absence or presence of the different bacterial extracts, and PMN adherence to HUVEC assessed 30 minutes later. Each value represents the mean + SE of 3 experiments performed in duplicate. *P < 0.01 and **P < 0.001 as compared with HBSS.

Figure 4. Effects of monoclonal antibodies (MAb) to CD1 la (TSl/ 22), CD1 1b (LM2/1), CD18 (184) ICAM- (R6.5), E-selectin (CL-2) P-selectin (G l), or mouse IgG nonbinding protein (NBP) on PMN adherence to HUVEC induced by the H. pylori extract. The MAbs were added along with the +Cr-labeled PMNs and the H. pylori extract (IO-fold dilution) to HUVEC monolayers and PMN adherence to HUVEC assessed 30 minutes later. Dashed line represents level of basal adherence observed in the absence of the H. pylori extract. Each value represents mean + SE of 3 experiments in triplicate. *P < 0.0 1 and **P < 0.001 as compared with untreated (no MAb) group.

1436

YOSHIDA ET AL.

GASTROENTEROLOGY Vol. 105, No. 5

PMN Pretreatment

CD18

CDlla

CDllb

Figure 6. Effects of H. pylori extract on surface expression of CD 1 la, CD1 1b, and CD 18 on neutrophils. PMNs were incubated with H. pylori extract (1 O-fold dilution) and MAb against CD 1 1a (TS l/22), CD 11 b (LM2/1), or CD 18 (IB4) followed by incubation with FITC conju-

HBSS

25 _

Extract

gated goat anti-mouse immunoglobulin. MAb binding to the surface of neutrophils was analyzed in a flow cytometer. Each value represents mean + SE of 3 experiments. *P < 0.001 when compared to controls , HBSS; LE, extract. incubated with HBSS (no MAb). ??

exposure to the H. pyloriextract to 3 hours also did not promote neutrophil-endothelial cell adhesive interactions (data not shown). To determine whether the extract-induced, CDI8-

HUVEC Pretreatment

dependent

increase

VEC was related

20 -

CD1 I/CDI8,

unlabelled

of

H. pyioti extract (Figure

The proadhesive be attributed

Extract

Figure 5. (A) Adherence of neutrophils pretreated with H. pylori extract to naive HUVEC. 51Cr-labeled PMN were incubated in H. pylori extract (1 O-fold dilution) for 30 minutes. After washing, the pretreated PMN were added to naive HUVEC and PMN adherence assessed 30 minutes later. Each value represents mean f SE of three experiments in triplicate. (6) Adherence of naive PMN to HUVEC monolayers pretreated with H. pylon extract. HUVEC monolayers were preincubated with H. pylori extract for 30 minutes. After washing, “Cr-labeled PMN were added to the pretreated HUVEC and PMN adherence assessed 30 minutes later. Each value represents mean 2 SE of 3 experiments in triplicate. *P < 0.01 as compared with HBSS pretreatment.

LTB4,

the extract-induced showed

of H. pylori extract or fMLP,

to each inflammatory

ure 7). Molecular

HBSS

surface

ex-

6).

activity

to PAF,

tor antagonists

proach

increased

of CD1 lb and CD1 8, but not CD1 la, on iso-

lated neutrophils

0

of

the H. pylon’extract for 30 minutes, and the extent MAb binding was assessed by immunofluorescence

prevent

neutrophils

to HU-

expression

to

pression

5

adherence surface

were exposed

flow cytometry.

10

in neutrophil

to an increased

sizing that

neutrophil analyses

because

mediator using

cannot recepdid not

adhesion a dialysis

(Figap-

13%, 34%, 49%, and 49% of the

adherence promoting activity of the H. pyLoriextract was lost across membranes with 10,000, 15,000, 25,000, and 50,000 mol wt cut off, respectively. Fractionation of the H. pyioti extract by agarose gel liltration confirmed that the proadhesive properties of the extract is attributable to multiple factors of different molecular weights. As shown in Figure 8, the three major peaks of proinflammatory activity were in fractions 20-23 (I-l.5 X IO6 mol wt), 36-39 (l-2 X IO5 mol wt), and 54-56 (~2-5 X lo4 mol wt). The fractions which promoted PMN adhesion to HUVEC (Figure 8A) also increased surface expression of CD1 lb/ CD18 on PMNs (Figure 8B). To further characterize

November

1993

H. PYLORI-INDUCED

30 r

NEUTROPHIL ADHESION

H. pylori extract

the entire

was exposed

tease, and acid treatments

1437

to heat, pro-

(data not shown).

Discussion A growing

body

pyLotiplays a pivotal and

duodenal

Ant

LTB

Ant

FMLP

+

acute

Figure 7. Role of inflammatory mediators in proadhesive effects of

is that H. pyloriinitiates

(WEB 2086)

inflammatory

mucosa.

a LTB,

dothelial tions

cells under

on fractions

23 and 55, which

sults in neutrophil

major

peaks

of activity

Figure

9, the proadhesive

was abolished

(see Figure activity

by heating

was not affected

by heating

The proadhesive

activity

with pronase

8). As shown

at 56°C

but was resistant

ship

that an

adhesion

and in vitro in vivo,

to encondi-

which

and accumulation

re-

in the

properties

did not exhibit

when

proadhesive

surprising

assessed

in

of H. fels, on

assay. An extract

did exhibit

in an

activity,

which

in view of the close relation-

H. fels and H. pylori4’ Furthermore, mice infected with H. fels exhibit gastritis

between

germ-free with

marked

histological

with pep-

sin. Acidification

did not alter the proadhesive

effect

human

of either fraction.

Identical

when

together,

results were obtained

leukocyte in vivo

an

gastrointestinal

to the H. pylon’ extract

adhesion

is not entirely

but

by treatment

to treatment

in the

response

proadhesive

the other hand,

in

for 30 minutes.

was abolished

reac-

H. pylori initiates

the first evidence

emigration

manner

our in vitro

to the two

for 10 minutes

an inflammatory

of E. coli and P. aemginosa prepared

significant

of the two fractions

at 100°C

exact

interstitium.

we focused

correspond

both

and a chemotactic

identical of the extract,

reaction

This study provides

Extracts properties

the

to the patho-

is not clear. One hy-

by which

of H. pyioripromotes

extract

receptor antagonist (SC 41930), or a fMLP receptor antagonist (N-tBoc-Met-Leu-Phe) were added along with the %r-labeled PMN and the H. pyloriextract (50-fold dilution) to HUVEC monolayers. Thirty minutes later, PMN adherence to HUVEC was assessed. Each value represents mean + SE of 3 experiments in triplicate. *P < 0.01 as compared with HBSS.

the proadhesive

ulcers

basis for the mechanism

Extract

the H. pylori extract. A PAF receptor antagonist

However,

of gastric

tion within the mucosa, which subsequently leads to tissue injury.‘,2,5 The results of this study provide a

Ant

I

I

ulcerations.‘-”

of gastroduodenal

pothesis PAF

HBSS

that H.

indicates

by which H. py/oricontributes

mechanism genesis

of evidence

role in the pathogenesis

neutrophil features

volunteers these

infiltration

reminiscent who

ingested

observations

of the mucosa,41

of those

observed

in

H. py/~ri.‘~*” Taken

indicate

that

the extrac-

.2 j

150-

c 8 s t e s G s f

IOO-

50-

“““““““““’ 10

20

21 22

23

27

30 3.3

36

39

49 54

Fraction Number

55

56

57 58

59 65

19

20

21

22

23

27

30

4Q

54

55

55

57

55

55

Fraction Number

Figure 8. The proinflammatory effects of various fractions (agarose gel chromatography) of the H. pylori extract on PMN adherence to HUVEC (A) and surface expression of CD 11 b/CD 18 on PMNs (6). Adhesion was assessed as described in legend of Figure 2 and PMN surface expression of CD1 la (r), CD1 lb (D), and CD18 (0) was assessed as described in legend of Figure 6. The dotted line in (A) represents basal neutrophil adherence. The three major peaks of proinflammatory activity were in fractions 20-23 (1- 1.5 X 1O6 rnd wt), 36-30 (l-2 X 1O5 mol wt), and 54-56 (2-5 X lo4 mol wt). Each value represents mean + SE of 3 experiments in duplicate or triplicate.

YOSHIDA

1438

g

ET AL.

GASTROENTEROLOGY

endothelial

100

course

ti

s

80

3 8 5 ii

in the H. pyiori extract-induced

20

playing

a role

extract,

this

cells more

0

Heat

Heat (WC,

Mm)

(100%.

Pepsin

Prcnase

HCL

that

for 3 hours

for neutrophils.

adhesion

used in the present that

appear

study yields proad-

fairly

specific

H. py/ori extract the selectins

is consistent

for H pylori

approach,

adhesive

along

the endothelium)

interactions,

than

attach

CD 18-ICAM-

Under

of activated

involves

both

eventually

neutrophils

mediator,

PAF receptor

to endothelial

urease

cells The

is that stimulation

MAb

or the combination

the inflammatory

in the present

of

strategy

in mediating

than of anti-

CD1 1 a and anti-CD1 1b MAbs. These findings suggest that CD 11 a and/or CD 11 b are also binding to ligands other than ICAMon endothelial cells in response to the H. pylon’extract. Our findings that MAbs directed against the E- and P-selectins did not diminish the hyperadhesive effect of the extract indicate that the selectins on endothelial cells do not play a significant role in PMN adherence to HUVEC induced by the extract. P-selectin is rapidly (within minutes) mobilized to the endothelial cell surface upon activation of these cells with inflammatory stimuli. However, several hours are required for maximal surface expression of the E-selectin on activated

that

extracts

led us to ex-

a molecular against

sive properties

weight

ef-

directly

assess whether

for the proadhesive However,

sizing experiments,

based

ef-

on our

at most only 50% of the

could

50% of the activity

Recent

urease2’ or lipopolysac-

(LPS).21 We did not

activity

the hy-

the proinflammatory

or LPS was responsible

molecular

a

of H. pyLori can be partially

to H. py/orz-derived

proadhesive

study,

did not affect the proadhesive A similar

fects of the H. py/ori extract.

ments

was less inhibitory

have

charide

MAb directed

the CD18-specific

antagonist

of the extract.

attributed

neutrophils with chemotactic agents leads to activation of CD 11 b/CD1 8 on neutrophils.22,24,30*42 The ICAM-

and secrete

However,

indicated

because

against

PAF.43

fects of certain

cells via a CDlla/

ex-

The identity of the proinflammatory factors in the H. pyiori extract is not clear. Others have reported that

studies

and CDllb/CD18.

view in the literature

lead to neutrophil

induced

conditions,

adhesive

travasation.24

by the H. pylon’ extract.

process.22,24,29 However,

CDlla/CD18

in the stronger

induced

and ICAM-

unstimulated

to endothelial dependent

adhesion prevailing

on neutrophils

role in the

of neutrophils

peradherence

on both CD1 1 a/

was dependent

important

(rolling

by the

view that

and fMLP

by the H. pylori extract CD18 and CDllb/CD18

more

interactions which

cells elicited

clude a role for LTB4

response

neutrophils

contention

we were

able to show that the hyperadhesive

cells.

Our

with the general

play a much

weak

activity

and its close relative, H. fels. Using an immunoneutralization

on endothelial

endothelial

the endothelial

do not play a role in the

to endothelial

H. pyloti can synthesize factors

by the

10m)

Figure 9. Effects of heat, protease, and acid treatment on the proadhesive activity of the two fractions of H. pylori extract exhibiting proinflammatory activity (fractions 23 [O] and 55 [a] in Figure 8). Details of the treatment regimens are given in the text. After the various treatments, the fractions were added along with 5LCr-labeled PMN to HUVEC monolayers and PMN adherence to HUVEC assessed 30 minutes later. Results are expressed as the percent of the adherence promoting activity of the untreated extracts. Each value represents mean f SE of 3 experiments in triplicate.

hesive

of the

the were

induced

have made

the E- and P-selectins

of endothe-

If E-selectin

exposure

should

ad-

did not render

for neutrophils.

adhesive

neutrophil

tion procedure

that exposure

prolonged

a

not the case is sup-

in the hyperadhesion

cells to the extract

Z

likely

by our observations

cells more adhesive

$40

precluded

is most

this

lial cells to the extract

.z i

assay (30 minutes)

role for E-selectin That

No. 5

it could be argued that the time

of our adhesion

hesion. ported

60

cells. Thus,

Vol. 105,

be due to urease is attributed

of 25,000

or LPS

to factors

or less. Further

with argu-

a role for urease or LPS in the proadheof our extract

of H. pylori are provided

by the following observations. In our molecular fractionation experiments, the fraction which contained the most urease (fraction no. 27) possessed negligible proinflammatory effects (Figure 8). With regards to LPS, pretreatment of endothelial cells with LPS for 2-4 hours renders them more adhesive for neutrophils. 22,24 In the present study, pretreatment of endothelial cells with the H. py/ori extract did not render the endothelial cell surface more adhesive for neutrophils. Our preliminary characterization of the H. pylon’ extract indicates that the proadhesive factor is heat labile and pronase sensitive, but acid and pepsin resistant. Further studies are warranted to identify the factor(s)

November 1993

in H. tory

pylori extracts

H. PYLORI-INDUCED

responsible

for

its proinflamma-

effects.

In summary, we have shown that a water extract of H. pyloti can promote neutrophil-endothelial cell adhesive interactions in vivo and in vitro. Some, as yet unidentified, cell surface material contained in the extract upregulates CD1 1b/CD1 8 on neutrophils and initiates a CDlla/CD18, CDllb/CD18-ICAMdependent PMN adherence to venular endothelium and subsequent emigration into the interstitium. These observations, as well as those of others,‘s-2’ support the contention that H. py/on’ infection can lead to an inflammatory response in the gastrointestinal mucosa, which ultimately results in mucosal injury. Opponents to this view argue that not all individuals who harbor the bacteria have gastrointestinal mucosal inflammation and associated pathology. One way to reconcile the disparate views is to assume that H. py/oti or H. pyLoti-derived inflammatory mediators do not penetrate an intact epithelial lining but can readily gain access to the mucosal interstitium and postcapillary venules if the barrier is disrupted by other factors such as stress, hyperacidity, etc. In support of the latter possibility is the recent observation that administration of H. pylori (or bacteria-free filtrates) produces gastritis only in those animals with a disrupted gastric mucosal barrier.44

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Received March 29, 1993. Accepted July 7, 1993. Address requests for reprints to: Peter R. Kvietys, Ph.D., Louisiana State University Medical Center, Department of Physiology, 1501 Kings Hlghway, P.O. Box 33932, Shreveport, LA 71130-3932. Supported by grants from the National Institutes of Health (DK 41399, DK 43785, DK 39919) and funds from the Department of Veterans Affairs.