Fibronectin

fragments

secretion

stimulate

by human

Donald

H.

Laboratory

necrosis

factor

monocytes

Beezhold

and

of Macrophage

tumor

Christine

Biology,

Personius

Guthrie

Research

Institute,

Abstract: Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatinSepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn-induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D-containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. Highperformance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cellbinding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.J. Leukoc. Biol. 51: 59-64; 1992.

Sayre,

for

lymph

the

lymph

Pennsylvania

node cells [22]. It was further demonstrated node cell proliferative response was Fn and macrophage dependent and that the were T lymphocytes [22]. recently, it has been shown that monocytes, and B lymphocytes express at least two

pendent ing cells More phocytes,

that dose derespondT lymintegrins

that function as Fn receptors, VLA-4 [12, 37] and VLA-S [24]. Fn-dependent proliferation of highly purified T cells is dependent on costimulation of the cells with anti-CD3 antibody and Fn [24, 33]. In light of these observations, it is postulated that the mitogenic activity of Fn in lymph node cell cultures involves costimulation of the T cells by Fn and macnophages. The exact nature of the involement of macrophages in this response remains to be determined. Monocyte/macrophages function as accessory cells by antigen processing and presentation. In addition, macrophages modulate the activity of other cell types by the secretion of several cytokines. We have previously shown that Fn can induce rat penitoneal macrophages to secrete intenleukin 1 (IL-i) [1]. We have now extended those studies to include the stimulation of TNF secretion by human monocytes, and we have localized the stimulatory activity to the cell-binding fragment of Fn. Thus, it may be hypothesized that Fn fragments are important mediators in host defense and function by the recruitment of monocytes and by activating the cells for increased effector cell function.

MATERIALS

AND

METHODS

Reagents Key

Words:

monocytes

jibronectin

tumor

necrosis

factor

Hepanin

and

gelatin-Sepharose 4B were purchased from (Piscataway, NJ). Chymotrypsin-agarose, polymyxin B, and Ficoll-Hypaque (Histopaque) were obtained from Sigma (St. Louis, MO). The synthetic peptides GRGDSP and GRADSP were purchased from Telios (San Diego, CA) and the anti-Fn antibody was purchased from Calbiochem (San Diego, CA). Pharmacia

INTRODUCTION Fibronectin

(Fn)

that

has

been

and

tissue

and

stimulate

injury,

sis

not

found

in

have of

We tory mixed

the

have activity

mitogens,

lymphocyte

collagen,

sensitive found including

metastatic

that

These for

dovanDNA,

tumor

various Fn

36]. fluids

as

a nonspecific

blood

Stimulation

of Monocytes

drawn

from

healthy

(10

U/mi)

and

hepanin

volunteers the

was

collected blood

peripheral

of

Fn fnagand at

Fn,

Abbreviations:

HPLC,

high-performance

lipopolysaccharide;

cells.

that Fn has immunomodulafound to inhibit the allogeneic [20] and nonspecific lymphocyte mitogens [2i}. In the absence

functioned

Venous

and

to pnoteoly-

to have activities stimulation [27,

Purification into

phosphate-buffered

shown Fn was response

to however,

fibnin,

is extremely

by

previously [20-23].

reactivity

molecule

of functional domains. by their binding affinity

[161 and chemotaxis identified in inflammatory

destruction

transformation

immune

is a complex

of Fn have been intact molecule,

been

tissue

Fn

of inflammation

sites

modulate

Fn

including

[30].

proliferation

ments sites

cells

glycoprotein at

it may repair.

molecules

fragments

fibnobiast

accumulate

by a series identified

been

and

and

high-molecular-weight

where

biological

hepanin,

a to

wound

is characterized mains have ous

is

shown

mitogen

sodium sis

dodecyl

HBSS,

Hanks’

chromatography; PBMC, peripheral blood saline; PEC, peritoneal exudate

sulfate-polyacrylamide

balanced

salt

solution;

!L-i , interleukin I ; LPS, mononuclear cell; PBS, macrophage; SDS-PAGE,

liquid

gel electrophoresis;

TNF,

tumor

necro-

factor. Reprint

of

fibronectin;

ogy,

requests:

Guthrie

Received

Journal

Donald

Research

March

of Leukocyte

H.

Institute,

26,

1991

Beezhold,

Laboratory

PA

Sayre, ; accepted

Biology

April

Volume

of Macrophage

Biol-

18840. 26,

1991.

51,

January

1992

59

mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll-Hypaque (i000g, 20 mm). The PBMCs were washed three times with Hanks’ balanced salt solution (HBSS) and resuspended in endotoxin-free RPMI 1640 (Cellgro; Mediatech, Herndon, VA) containing 100 U/mi penicillin, 100 jg/ml streptomycin, 20 mM HEPES, and 10% fetal bovine serum. One milliliter of the cell suspension (2 x 106/well) was plated into 24-well tissue culture plates (Linbro; Flow Laboratories, McLean, VA) and allowed to adhere for 2 h at 37#{176}C.Nonadherent cells were removed by vigorous washing three times with HBSS. The remaining ad-

against PBS, and protein concentrations optical density at 280 nm using an 1.28 mg/ml [26].

herent cells (2 x i05) were 93-95% monocytes as determined by Giemsa staining and the phagocytosis of latex beads (1 jim; Dow Diagnostics, Indianapolis, IN). The monocytes were stimulated with Fn or Fn fragments in serum-free RPMI for i8 h, after which the supernatants were collected, centrifuged (iO,000g, 4 mm) and assayed for

extent of fragmentation sulfate-polyacrylamide

tumor

necrosis

Peritoneal

factor

(TNF)

activity.

Macrophages

Mice of the C3H/Hej (endotoxin-resistant) and C3H/Ouj ( endotoxin-sensitive) strains were purchased from Jackson Laboratories (Bar Harbor, ME). Periotoneal exudate macrophages (PECs) were recovered from the mice 3 days after intraperitoneal injection of i.0 ml of a sterile 10% solution of thioglycoliate broth (Difco, Detroit, MI) as previously described [2]. The exudate cells were plated at 1 x i06/ml and allowed to adhere for i h before vigorous washing to remove

nonadherent

cells.

Fibronectin

Proteolysis

Fibronectin

fragments

procedure with insoluble equivalent zyme

10%

of Fibronectin

Fibronectin was purified from plasma by gelatin-Sepharose chromatography as previously described [22]. In brief, human plasma was batch incubated with gelatin-Sepharose for 2 h at room temperature. The slurry was poured into a column and washed extensively with phosphate-buffered saline (PBS), ph 7.4, followed by 0.1 M urea in S mM Tris-HC1 buffer, pH 7.5. Fn was eiuted with 4 M urea in the same Tris buffer and dialyzed against PBS containing 100 U/ml penicillin, 100 tg/ml streptomycin, and 0.1 zg/ml polymyxin B. The Fn was concentrated by negative pressure dialysis

60 55

5 units

removed

gel.

The

stimulating

produced

by

by

chymotrypsin

per

centrifugation

(l2,000g,

fragments

were

or

mg

tested

further

purified

by of

a modification

et al. [26]. Fn (chymotrypsin-agarose)

was determined gel electrophoresis

activity

determined coefficient

was of

of

incubated at

Fn.

The

an en-

2 mm) by sodium (SDS-PAGE)

and the dodecyl on a directly for TNF by affinity chro-

matography.

For heparin-Sepharose affinity chromatography, the fragments were first dialyzed against 25 mM Tris buffer, pH 7.5, and then batch incubated with heparin-Sepharose for i h. The fragments were eluted in two fractions in the same Tris buffer

containing

tion

200

was

again subjected and the gelatin-binding collected, dialyzed concentrated. The

phy

tions and

mM

NaCl

and

column column.

cyte

fragment

stimulation

treated

with contaminating

lized man In ther phy

iM

NaCl.

Each

frac-

to gelatin-Sepharose chromatograand non-gelatin-binding fracagainst PBS containing antibiotics, 120-kd cell-binding fragment was

found to elute from the heparin yet failed to bind to the gelatin

any

Purification

of was

were

of Pierschbacher chymotrypsin

the

were extinction

assays,

Detoxi-Gel

all

(Pierce, lipopolysaccharide through a 0.2-jim

with 200 mM NaC1, Before use in monopreparations

were

Rockford, IL) to eliminate (LPS) and filter steripore filter (Acrodisc, Gel-

by passage Sciences). some experiments, the fragment preparations were furfractionated by high-performance liquid chromatogra(HPLC) on a GF-2S0 zirconia-stabilized silica gel filtration column (Du Pont). The mobile phase was PBS at pH 7.4 and the flow rate was 1.0 mi/mm. Following sample injection and an 8-mm wait, the fragments were collected in 0.5-mi or 0.25-mi fractions and analyzed for TNF stimulatory activity using

human

were

identified

sis

as

previously

peripheral

in the HPLC described

blood monocytes. Fn fractions by western [35].

fragments blot analy-

lb

Ia

I

50

45 40 35

30

30

25

20

20 15

10

10 5 a

Con

10

1.0 Fn

(ug/ml)

R-G-D

(ug/ml)

Fig. 1. Stimulation ofTNF secretion by intact plasma fibronectin. (a) Dose response ofadherence-purified monocytes to Fn. The monocytes were stimulated with gelatin-Sepharose-purified plasma Fn for 18 h in serum-free medium and the supernatant levels of TNF determined by the L929 bioassay. The data are representative of six different Fn preparations. (b) Inhibition of Fn-induced TNF secretion by R-G-D peptide. Adherence-purified human monocytes were stimulated with plasma FN (100 zg/ml). The R-G-D-containing peptide GRGDSP was added 30 mm prior to the addition of Fn and the supernatants were collected after 18 h incubation. The peptide inhibited Fn-induced TNF secretion in a dose-dependent manner. No inhibition was observed with the control

60

peptide.

Journal

of Leukocyte

Biology

Volume

51,

January

1992

Tumor

Necrosis

The L929

Factor

biological fibroblast

[3].

Units

activity of TNF was tested using cytotoxicity assay [11] as previously

of

interim

activity

were

recombinant

tional

determined

TNF

Biological

the

as

to

from

distributed

Program,

the

by

National

MD. Units of activity calculated using Parlin

murine described

comparison

obtained

Board

Modifiers

tute, Frederick, intervals were

by

standard

Standards

Response

logical

MW

Bioassay

0

5

10

30

60

25 the

Na-

the

Bio-

Cancer

FN

12

Insti-

9

and 95% confidence software [17].

6 RESULTS Fibronectin Intact

Stimulation

Fn

was

the

tants.

TNF

relatively

needed

high

to

induce

serum-free and

by

an

inhibit

to demonstrate neutralize

R-G-D

sequence

binding TNF

in Figure

inhibited

in

been

function

of

system,

Fn-induced

the

TNF

In

3. SDS-PAGE

Fibronectin reducing

cell-

staining. above

to

the

after

secretion

in

a dose-

response.

As

a

first

molecule

step

in

was

fragmented

con

Fn

by Fn Fragments

determining

the

by

active

sites

limited

the

labeled

the

level

of

the

production

addition

on

proteins

with

lane

Figure

fragmentation

of

polyacrylamide

visualized

chymotrypsin Fn

by chymotrypsin.

a 10%

by

(in

represents

minutes)

induced

Blue

is

untreated

fragmentation

gel

Coomassie

indicated

monomeric

by

Fn,

chymotrypsin

im-

of chymotrypsin.

ability

to

in

Fn,

proteolysis

the

the

the

indicated

times.

compared

to

the

Fn

TNF

and

with

insoluble

chymotrypsin-agarose

removed

by

centrifugation at of the samples prior to the addition

TNF-inducing

intact

the

enzyme.

Fn

activity

Fn This

in

these

In

was

analysis that an increased experiments, Fn

results

insoluble

enzyme

between

by kinetic

molecule

secretion.

incubated

insoluble

sampled approach

preparations

eliminates

[2,

23]

or

the

in the

varia-

of proteolysis. The extent of fragmentation was analyzed by SDSPAGE (Fig. 3). Although a large number of fragments are produced, accumulation of a i20-kd fragment is observed over

using

induce

batch

was

2 demonstrates the

was

bility

Secretion

treatment

chymotrypsin.

of

of TNF

and

The

0 represents

fragment

electrophoresed

peptide

R-G-D-contain-

dependent manner. The control peptide GRADSP had no effect on Fn-induced TNF secretion (data not shown). However, complete inhibition could not be obtained using the peptide GRGDSP, suggesting that different active sites on the molecule or multiple Fn receptors are involved in this

Stimulation

of

lane.

mediately

order

of Fn

were

conditons

Time each

and

analysis

fragments

under

pep-

shown

Fn.

Fig.

we attempted

using

ib,

the small

have

secretion

in

control in this to bind to many

known

in our

are

cultured

Furthermore,

and

Fn

can

be seen

dependent,

10 tg/ml)

contained

[31).

Fn-induced

As

peptide

is

sequence

for

dose

than

as a negative

molecule the

Fn

by human Fn stimusuperna-

Monocytes Fn

specificity

GRGDSP.

was

(greater

served

R-G-D

the

specifically

ing

secretion.

ofthe

containing

to

TNF alone

of

region

tides

of Fn

experiments.

way

binding

secretion

doses

medium

subsequent

cells

4

found

Fn-induced

but

Monocytes

to induce cytokine secretion As can be seen in Figure la, plasma secretion ofTNF activity in the monocyte

monocytes.

lated

of Human

rate

time.

30 25 2O C

15

: Li

5.

5

0

10

Chymotrypsin (in Fig.

2.

Kinetics

Plasma

Fn

Sigma)

and

of Fn

was

treated

ments

for

assay.

Cells

representative

with

sampled

stopped by removal was then determined 18 h and were of

fragment

at

insoluble

the

of the by

production

enzyme incubating

supernatant stimulated three

different

with TNF

at

times.

50

levels

fragment

1

2

3

4

treatment.

enzymatic

were

assayed or

preparations.

Fn

digestion

Stimulatory with the with fragments.

the

5

6

7

Fraction

(chymotrypsin-agarose, The

Fn

0

chymotrypsin

centrifugation. human monocytes

g/ml

60

mm)

by

chymotrypsin

indicated

30 digestion

was activity Fn frag-

Fig.

4.

Monocyte

ments.

Chymotryptic

GF-250

gel

L929

bio-

TNF-inducing

Data

are

of

TNF

three

Beezhold

TNF

filtration activity

and

column. in the activity

fragment

Personius

of The

Fn

(1

by

observed.

HPLC-separated

mg/mI)

fractions

monocyte are

91011121314

number

induced

fragments

stimulatory

different

secretion

8

(10

were l)

stimulation The

were assay.

data

Fn

fractionated then Two

are

fragon

tested major

representative

a for

peaks of

preparations.

Fibronectin-induced

TNF

secretion

61

b

a

MW

C

Western

blot

several

analysis

bands

of peak

including

lower-molecular-weight

TNF-lnducing

Activity

6-8

(fraction

the

i20-kd

bands

(Fig.

7) demonstrated

fragment

and

several

5).

Is Not Endotoxin

Contamination

The possibility of endotoxin contamination must counted for in all experiments that examine cytokine tion. Therefore, we have examined the stimulatory of

Fn

fragments

using

were

performed

described The

was

using

in relation

tions.

fragment

fraction that addition,

fractions

to

the

used

as

(200 contains the the fragments

further

following

As

gesting

starting

discriminate

the

Fig.

5.

Western

chymotryptic l20-kd

blot digest

of Fn.

cell-binding

contains

HPLC

of Fn

Lane

fragment.

multiple

represents

analysis

a represents Lane

fragments

HPLC

fraction

Purification

fragments

in

HPLC

HPLC

b represents

including

fractions

fraction HPLC

the

3, which fraction

l20-kd

of the

not

is the 7,

fragment.

c

10.

activation

the

response

a requirement

for

macrophages

which

Lane

macrophage

augment

from

to

the

However,

Fn-induced

C3H/Ouj

Fn fragments in GRGDSP completely had no effect (Fig.

(perhaps Fn.

TNF

mice

the presence eliminated 7).

material

were

peaks

of

ac-

from both cells secrete shift in response the result of colfractions during C3H/Ouj cells C3H/Hej, sug-

can

that

may

as

modifica-

mM NaCl), gelatini20-kd cell-binding were collected in

be seen in Figure 6, macrophages C3H/Hej and the endotoxin-sensitive C3H/Ouj TNF in response to HPLC fraction 5. The to fraction 5 from fractions 2-3 (Fig. 5) was lecting 0.25-mi fractions instead of 0.5-mi the HPLC purification. Interestingly, the respond more strongly to Fn than cells from tivity.

experiments fragments

4 with

preparation

from

These

HPLC-purified

heparin-binding

In

0.25-mi

macrophages

mice.

to Figure

a low-affinity

nonbinding fragment.

peritoneal

C3H/Hej

endotoxin-insensitive

be acsecreactivity

by

endotoxin)

LPS

is

clearly

secretion.

When

stimulated

with

of synthetic the response and

the

peptides, GRADSP

of Fn Fragments DISCUSSION

Chymotrypsin treatment of Fn results in the production of a large number of proteolytic fragments. To isolate the stimulator)’ regions of the Fn molecule, the fragments were separated by HPLC gel filtration chromatography and tested for TNF stimulatory activity. As can be seen in Figure 4, two peaks of TNF stimulatory activity were observed. Western blot analysis (Fig. 5) of the first peak (fraction 3) reveals that the major fragment present is the i20-kd fragment of Fn. This fragment comigrates with the 120-kd cell-binding fragment purchased from Calbiochem (data not shown).

In

this

of

a

study

new

we confirm

biological

and

function

extend

our

of

stimulation

Fn:

previous

finding of

[1]

cytokine

secretion. In this study we have shown that purified plasma Fn is capable of stimulating TNF release from adherencepurified human monocytes. The cytokine-inducing activity of the intact molecule was partially blocked by GRGDSP

600

500 400 400 (1) 4-’

C

300

300

E (1) 4-,

LL

z

C

200

I-

100

0

2

1

3

4

HPLC 00

1

2

Fig.

6. Fn-induced

TNF

tion.

Macrophages

from

3

4

HPLC

Fraction

secretion

is not

both

C3H/Hej

line) mice secrete TNF in response The C3H/Hej macrophages failed up to 10 /g/ml (data not shown).

62

Journal

5

of Leukocyte

6

(dashed

Biology

8

9

10

by endotoxin line)

fraction to LPS

Volume

Fig.

7.

Inhibition

of

Fn

fragment-induced

thetic peptide. Data were obtained stimulated with 10 zl of HPLC-purified

number caused

to HPLC to respond

7

and

contaminaC3H/Ouj

thetic

(solid

5 of the Fn fragments. when stimulated with

51

,

January

1992

tion

peptides ofthe

Fn

(50 zg/ml) fragments.

5

were No

6

Fraction

added inhibition

TNF using

7

secretion

macrophages

from

Fn fragments

(solid

to the ofFn

8

9

by

R-G-D

10

Number

assay

30 mm

fragment-induced

C3H/Ouj

line). prior

synmice

The

syn-

to the

addi-

TNF

secre-

tion was observed when the control synthetic peptide GRADSP (dashed line) was added. However, the Fn inhibitor synthetic peptide GRGDSP (dotted line) completely inhibited this response.

and was dramatically augmented of the Fn. HPLC purification demonstrated that TNF-inducing least

two

i20-kd i20-kd

ofthe

Fn,

proteolytic

fragment. fragment

[26].

Chymotryptic that contains

by chymotryptic digestion of the chymotryptic fragments activity is located on at of Fn, one of which is a digestion of Fn produces a the central cell-binding region

Unlike

fragments

that

of

the

cell-binding

the

synthetic GRGDSP. Several observations

tory

activity

intact

fragment

in

our

isolated

and

was

in

activity eliminated

that

with

to

buffers polymyxin

the

by

stimulato conwe have taken rigorous precautions to in our Fn preparations. Our Fn is due

endotoxin-free

absorbed

TNF-inducing

completely

demonstrate

preparations

taminating LPS. First, reduce LPS contamination was

the

was

Fn

TNF

and

not

containing

antibiotics

B-Sepharose

before

use

in

our assays. Second, using affinity chromatography and HPLC we have localized Fn stimulatory activity to a fraction containing the i20-kd fragment. It is unlikely that LPS would coelute with this fragment and be inhibited by the GRGDSP peptide, which is known to block Fn binding to VLA-5. Finally, macrophages from endotoxin-insensitive mice (C3H/Hej) respond to the same HPLC fraction (fraction 5) of the Fn fragments. What is not known, however, is whether

LPS

serves

as

an

important

costimulator

in

our

as-

says. The fact that the cells from C3H/Ouj mice respond more strongly than C3H/Hej cells argues that LPS may enhance the response. In support ofthis, it has been shown that LPS enhances monocyte adherence to fibronectin [29]. Experiments are in progress to address this possibility. Monocytes are known to have at least two Fn receptors, VLA-4 and VLA-5 [4, 10], and are reported to interact with T cell Fn by way of a fucose receptor [13]. The mechanism by

which

Fn

stimulates

monocytes

to

secrete

TNF

is

not

known

but is likely to involve multiple interactions. Many including monocytes use Fn as a substrate adhesion molecule, yet it is now clear that interaction with Fn also alters the physiological activity of the cell. Monocytes have

cells

clearly

been

ments.

shown

Soluble

phages

to Fn

[i4,iS],

[25,

28]. Fn monocytes

macrophages stimulating but

soluble

Fn

shown

to

the

growth monocyte/macrophage

and/or

secretion

factor

Fn

agglutinate of

[23] and cytotoxic

frag-

macromono-

IL-i [1], activity

fragments have been shown to be chemotactic [7] and to enhance complement-mediated [8]. Thorens et al. [34] have shown that murine express granulocyte-macrophage colonyfactor mRNA after adherence to Fn-coated

phagocytosis

plates

to

been

stimulate

cyte/macrophage-derived and to augment for

respond

has

not

on

exposure

of

nonadherent

capable of fragmenting Fn [5, 32]. The Fn fragments may serve as a chemoattractant to direct monocytes to the tissue sites. At these tissue sites, Fn or Fn fragments may further activate monocytes to stimulate wound repair or tumor cell destruction. Furthermore, Fn can also serve as an adhesion [12, 37] and costimulatory molecule for T lymphocytes [24, 33]. Thus, Fn bound to the monocyte surface may serve as an important cell-cell adhesion molecule [18] and cytokineinducing signal that augments lymphocyte reactivity.

peritoneal

cells

ACKNOWLEDGMENTS The authors assistance.

1. Beezhold,

or

Fn

enhances

the

expression

of

IL-i

and

[9]

are

not

contradictory;

rather,

taken

together

they

that

Fn

can

have

variable

effects

Lause,

for

D.B. by

Invest. 16, 437, 1987. 2. Beezhold, D.H., Best, G.K., M. Endotoxin enhancement i-induced

secretion

Rev. Infect. 3. Beezhold,

tumor

his

excellent

E.J.,

and

technical

Med.

167,

the

of

P.F., shock

1 by

and Thompson, syndrome toxin macrophages.

J.A.,

and

Hall,

secretion

RE.

Fibronectin

RAE,

Dvorak,

HF.,

and

receptors

126, 787, 1981. 7. Clark, RAE, Wilkner,

N.E.,

of phagoproteins j

RB.

D.E.,

from

Fibronectin

reactions: associations cell activation. j

Doherty,

of

Exp.

proteases 193, 1987.

48,

Colvin,

skin endothelial

induces

monocytes.

binding leukocytes.

77, 1988. and Chen, W. Fibronectin-degrading membranes of transformed cells. Cell

delayed-type hypersensitivity vessel permeability and

P48

by human

J. ,

6. Clark,

macroImrnunol.

murine

of the Arg-Gly-Asp polymorphonuclear

and

rat

fibronectin.

1989.

Goodwin,J.L.

Characterization monocytes

5. Chen,

interleukin

S289,

Leftwich,

cytes. human

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