Proteinases in inflammation and tumor invasion: Review articles including those from an international conference, Bielefeld, Federal Republic of Germany March 14–16, 1985 9783111520049

Table of contents :
PREFACE
CONTENTS
GRANULOCYTE PROTEINASES AS MEDIATORS OF UNSPECIFIC PROTEOLYSIS IN INFLAMMATION: A REVIEW
EFFECT OF GRANULOCYTE ELASTASE ON THE METABOLISM OF PROTEOGLYCANS IN INFLAMMATORY JOINT DISEASES
PROTEINASE INHIBITOR THERAPY OF SEVERE INFLAMMATION IN PIGS: FIRST RESULTS WITH EGLIN, A POTENT INHIBITOR OF GRANULOCYTE ELASTASE AND CATHEPSIN G
MAST CELLS AND MATRIX DEGRADATION IN THE RHEUMATOID JOINT
HUMAN CATHEPSIN B, H AND L AND THEIR ENDOGENOUS PROTEIN INHIBITORS : STRUCTURE, FUNCTION AND BIOLOGICAL ROLE
CATHEPSIN B: A PROTEINASE LINKED TO METASTASIS?
THE COLLAGENS - MOLECULAR AND MACROMOLECULAR STRUCTURES
PROTEOLYTIC MODIFICATIONS OF TYPE VI COLLAGEN
DEGRADATION OF SPECIFIC COLLAGENS BY MACROPHAGES AND NEUTROPHILS
CONNECTIVE TISSUE METALLOPROTEINASES AND MATRIX DEGRADATION
TISSUE INHIBITOR OF METALLOPROTEINASES
COLLAGEN DEGRADATION IN PERIODONTAL DISEASES
INTERSTITIAL COLLAGENASE, GELATINASE AND A SPECIFIC TYPE IV/V (BASEMENT MEMBRANE)COLLAGEN DEGRADING PROTEINASE FROM HUMAN LEUKOCYTES
THE ROLE OF CELL MOTILITY IN TUMOR INVASION
PERITUMORAL EXTRACELLULAR MATRIX DEGRADATION IN THE LOOSE CONNECTIVE TISSUE OF THE MESENTERY
PLASMINOGEN ACTIVATORS AND TUMOR INVASION
THE ROLE OF PROTEINASE INHIBITORS IN CELLULAR INVASIVENESS: Purification and characterization of a serine proteinase inhibitor from rat chondrosarcoma
CHARACTERIZATION OF A COLLAGENASE INHIBITOR FROM HUMAN AMNIOTIC FLUID WHICH INHIBITS TUMOR CELL INVASION
DEVELOPMENT OF A DIRECT MEASUREMENT ASSAY FOR COLLAGENASE AGAINST TYPE I AND TYPE IV COLLAGENS IN TISSUE HOMOGENATE AND ITS APPLICATION IN STOMACH AND LUNG CANCERS
METASTATIC ABILITY OF LEWIS LUNG CARCINOMA CELLS AND EXTRACELLULAR PROTEOLYSIS. INTERACTIONS BETWEEN TUMOUR CELLS AND HOST MACROPHAGES IN THE DEGRADATION OF TYPE I AND TYPE IV COLLAGEN
A T-LYMPHOMA DERIVED PROTEINASE, SYNERGIZING WITH AN ENDOGLYCOSIDASE IN THE DEGRADATION OF SULPHATED PROTEOGLYCANS IN SUBENDOTHELIAL EXTRACELLULAR MATRIX
PROTEOLYTIC ACTIVITY IN CULTURED HUMAN LUNG CANCER CELLS
LIST OF AUTHORS
SUBJECT INDEX

Citation preview

Proteinases in Inflammation and Tumor Invasion

Proteinases in Inflammation and Tumor Invasion Review Articles

including those from an International Conference Bielefeld, Federal Republic of Germany March 14-16,1985 Editor Harald Tschesche

W G DE

Walter de Gruyter • Berlin • New York 1986

Editor

Harald Tschesche, Dr. rer. nat. Professor für Biochemie Lehrstuhl für Biochemie Fakultät für Chemie Universität Bielefeld D-4800 Bielefeld Federal Republic of Germany

Library of Congress Cataloging in Publication Data Proteinases in inflammation and tumor invasion Includes bibliographies and index. 1. Inflammation—Congresses. 2. Proteinase—Physiological effect—Congresses. Cancer invasiveness— Congresses. 4. Collagen diseases—Congresses. I. Tschesche, Harald. [DNLM: 1. Inflammation—congresses. 2. Neoplasm Invasiveness—congresses. 3. Peptide Peptidohydrolases—congresses. QZ 202 P967 1985] RB131.P77 1986 616'.0473 86-16708 ISBN 0-89925-216-8 (U.S.)

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Proteinases in inflammation and tumor invasion : review articles including those from an international conference, Bielefeld, Fed. Republic of Germany, March 14-16,1985 / ed. Harald Tschesche. - Berlin ; New York : de Gruyter, 1986. ISBN 3-11-010530-6 NE: Tschesche, Harald [Hrsg.]

Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.

PREFACE The

central

invading

that

the

last

are

also

as

into

ticemia,

in m a n y

features

decade and

tures

have

the of

these

same

the

It will and

in t h e the that

period

is of

become

host

patho-

matrix

such

and

o n e of t h e m o s t

host the

states,

cirrhosis

has

and

and

as

is a p r e r e q u i s i t e

subject

But

sep-

cancer.

conspicuous

structures

cells.

the

severe

enzymes

normal

role

in

during

tissue

disease

invasion

and

deleterious

has

only

for

been

attri-

in t h e

of w i d e s p r e a d

knowledge

of t h e m a i n

matrices,

e.g.

the

natural

the

plasma

the

aim of

inhibitors

various

important

and

substrates

cellular

collagenase,

n o t all

and

The

our

contribution

specific

organs

tumor

for

intramaterial

demonstrated

in p l a s m a

arthritis,

destruction

enzymes

isolated

be

and

invasion,

Numerous

antagonists, been

pathological

in

interstitial

various

be r e s p o n s i b l e

cells.

and

extracellular

as

cathepsins,

by

cell

accumulated

only

of p r o t e o l y t i c

triggered

rheumatoid

the

been

or

has

not

plasma. has

of

against

respective

phagocytized

tissue

tissue

release

study

the

roles

defense

last

interest

investigation.

improved.

have

to

by

incorporated,

circulating

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severe

of

important

proteins

host

evidence

surrounding

on

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intensive

produced

surrounding

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enzymes

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The

specific

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into

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processes.

to

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buted

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proteinases

inflammatory logical

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microenvironment shown

system

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released

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phagocytic

enzymes

protein

leukocyte

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proteolytic

fluid of

and

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cellular but

of

microorganisms

functions Within

role

plasminogen and

of

hydrolases,

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activator

intracellular

and

fea-

elastin, -

their

proteinase

pro-

has

great-

elastase, natural

inhibitors

-

characterized.

of f u t u r e

research

individual towards

pathological areas

of t h e s e

(leukocyte)

structural

collagen,

in t h i s

the more

the

to e l u c i d a t e t h e

enzymes goal

of

conditions. field.

recent

The

aspects

and

their

understanding This editor could

book

significance

interplay their

reviews

is a w a r e

with

of

be c o v e r e d ,

role

some the

of fact

since

the

VI book

evolved

from

a conference

1985

- 16,

at

March

14

(ZIF)

in B i e l e f e l d .

close

interdiscipiinary

book

members of

will of

This

interested might

The meeting

be of

tissue

volume

to

decreasing

also

explore

or

between

held

from Research

framework

scientists

I would

to

express

like

have

stimulated,

endeavor

financial Technology

would

support and

for

from

further

tissue

Darmstadt

my

Fonds

(ZIF),

Bielefeld. and

to m y

engagement

and

help

of

Bielefeld,

April

help

1986

inva-

chemists

in g e n e r a l and

and pharma-

approaches

protein

for

destruction

the

appreciation supported

possible

Federal from:

in

for

indebted

Werke, Chemie

to

the

organisation

of

staff

and the

Harald

those

generous

Research

and

I am a l s o

Bielefeld; AG, of

grateBayer

Hannover;

Interdisciplinary

assistents

all

conference.

the

of

Industry.

Kali

to

the

without

Ministry

Asta

Frankfurt;

Center

also

and

the Chemical

technical in

cancer

protein

therapeutic

to a n d

been

German

AG,

the

I am

Dr. Wenzel

and

phenomena

oncologists

plasma

gratitude

have

the

and

for

and

for

breakdown

clinical

the

and with

a basis

tissue

prospects

financial

Merck,

of and

biochemists

with

invasion.

not

AG, Wuppertal ; Degussa

biologists,

provide

contributed

of

the

to all

breakdown

inhibiting tumor

for

a unique

and m a t r i x might

the

and

ful

provided

confronted

experimental

inflammation

This

subject,

Interdisciplinary

profession

in the m e c h a n i s m s

encourage

cologists

who

same

for

cooperation

interest

the m e d i c a l

inflammatory

sion.

on t h e

Center

disciplines.

various

The

the

Research the Z I F ,

students

for

conference.

Tschesche

to

their

CONTENTS Granulocyte Proteolysis

P r o t e i n a s e s as M e d i a t o r s of in I n f l a m m a t i o n : A R e v i e w

H. F r i t z , M. J o c h u m , K . H . D u s w a l d , H. K o r t m a n n , S. N e u m a n n , H . L a n g

H.

Unspecific

Dittmer, 1

E f f e c t of G r a n u l o c y t e E l a s t a s e on the M e t a b o l i s m P r o t e o g l y c a n s in I n f l a m m a t o r y J o i n t D i s e a s e s K.

Kleesiek,

R.

Reinards,

of

H. G r e i l i n g

25

P r o t e i n a s e I n h i b i t o r T h e r a p y of S e v e r e I n f l a m m a t i o n in P i g s : F i r s t R e s u l t s w i t h E g l i n , a P o t e n t I n h i b i t o r of G r a n u l o c y t e E l a s t a s e and C a t h e p s i n G M. J o c h u m ,

H.F.

Welter,

Mast

Cells

and

D.E.

Wool 1 e y , J . R .

Matrix

M. S i e b e c k ,

Degradation

Yoffe,

J.M.

H.

in

Fritz

the

53

Rheumatoid

Joint

Evanson

61

Human C a t h e p s i n B, H and L and their E n d o g e n o u s P r o t e i n I n h i b i t o r s : S t r u c t u r e , F u n c t i o n and B i o l o g i c a l Role V. T u r k , J. B r z i n , M . K o p i t a r , M. K o t n i k , B. L e n a r c i c , T . P o p o v i c , A. R i t o n j a , M. T r s t e n j a k , B. R o z m a n , W. M a c h l e i d t

Cathepsin B.F. J.D. The K.

B: A P r o t e i n a s e

to

Collagens

- Molecular

and

Metastasis? T.T.

Lah, 93

Macromolecular

Structures

Kühn

Proteolytic J.

Linked

S l o a n e , R . E . R y a n , J. R o z h i n , C r i s s m a n , K.V. Honn

107

Modifications

Rauterberg,

Degradation Neutrophi1s C.L.

77

R. J a n d e r ,

of S p e c i f i c

Mainardi,

M.S.

of T y p e

Connective

Tissue

G. M u r p h y ,

J. G a v r i l o v i c ,

Collagen

D. T r o y e r

Collagens

Hibbs,

VI

145

by M a c r o p h a g e s

and

K. A. H a s t y

Metalloproteinases Ch.

161 and

McAlpine

Matrix

Degradation 173

VIII Tissue T.E.

Inhibitor

Cawston,

Collagen V.-J.

E.

of

Metalloproteinases

Mercer,

Degradation

in

C.

Mooney,

J.K.

Periodontal

Wriaht

189

Diseases

Uitto

211

I n t e r s t i t i a l C o l l a g e n a s e , G e l a t i n a s e and a S p e c i f i c Type IV/V ( B a s e m e n t Membrane) C o l l a g e n D e g r a d i n q P r o t e i n a s e f r o m Human L e u k o c y t e s H. T s c h e s c h e , J. M i c h a e l i s , The

Role

of

J. K.

Cell

F e d r o w i t z , U. K o h n e r t , K ü h n , H. W i e d e m a n n Motility

in

Tumor

H.W.

Macartney,

Invasion 245

G. Haemmerl i Peritumoral E x t r a c el l u l a r Matrix Degradation Loose C o n n e c t i v e T i s s u e of the Mesentery

in

the

W. M u l 1 e r - G l a u s e r

261

Plasminogen

Activators

a n d Tumor

L. K.

L.R.

P.A.

Skriver, DanizS

Lund,

Invasion

Andreasen,

Levy,

G.

Kristensen, 281

The R o l e o f P r o t e i n a s e I n h i b i t o r s i n P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f I n h i b i t o r from Rat Chondrosarcoma H.

P.

Cellular a Serine

Invasiveness: Proteinase

Feinstein

C h a r a c t e r i z a t i o n o f a C o l l a g e n a s e I n h i b i t o r f r o m Human A m n i o t i c F l u i d w h i c h I n h i b i t s Tumor C e l l Invasion H.

Levy,

P.

Mignatti,

D.B.

Rifkin

Development of a D i r e c t Measurement Assay f o r C o l l a g e n a s e a g a i n s t T y p e I and T y p e IV C o l l a q e n s i n T i s s u e H o m o g e n a t e and i t s A p p l i c a t i o n i n S t o m a c h and L u n g C a n c e r s K.

Kubochi,

K.

Maruyama,

I.

Okazaki

M e t a s t a t i c A b i l i t y o f L e w i s Lung C a r c i n o m a C e l l s and E x t r a c e l l u l a r P r o t e o l y s i s . I n t e r a c t i o n s b e t w e e n Tumour C e l l s and H o s t M a c r o p h a g e s i n the D e g r a d a t i o n o f Type I and T y p e I V C o l l a g e n G.

Vaes,

N.

Henry,

A.L.

van

Lamsweerde,

Y.

Eeckhout

...

IX

A T - L y m p h o m a D e r i v e d P r o t e i n a s e , S y n e r g i z i n g w i t h an E n d o g l y c o s i d a s e in t h e D e g r a d a t i o n of S u l p h a t e d P r o t e o g l y c a n s in S u b e n d o t h e l i a l E x t r a c e l l u l a r M a t r i x M.D. Kramer, I. VI o d a v s k y Proteolytic

V.

Schirrmacher,

Bar-Ner, 373

Activity

O.L.

Schoenberger,

H.O.

Werling,

List

of A u t h o r s

Subject

M.

Index

in C u l t u r e d

Human

H. S c h w ö b e l , W.

N. P a w e l e t z

Lung

Ebert,

P.

Cancer

Cells

Aulenbacher, 395 409 411

GRANULOCYTE PROTEINASES AS MEDIATORS OF UNSPECIFIC PROTEOLYSIS IN INFLAMMATION: A REVIEW

Hans Fritz, M.Jochum, Department of Clinical Chemistry and Clinical Biochemistry, University of Munich, Munich, FRG.

K.H. Duswald, H. Dittmer, H. Kortmann Surgical Clinic City and Surgical Clinic GroBhadern, University of Munich, Munich, FRG.

S. Neumann and H. Lang Biochemical Research Department, E. Merck, Darmstadt, FRG.

Abstract

In severe inflammatory response, various blood and tissue cells, including polymorphonuclear granulocytes, release lysosomal proteinases, extracellularly and into the circulation. Such enzymes, as well as normally intracellular oxidizing agents produced during phagocytosis, enhance the inflammatory response by degrading connective tissue structures, membrane constituents and soluble proteins by proteolysis or oxidation. We first used polymorphonuclear elastase (E) as a marker of such release reactions. The liberated proteinase competes with susceptible substrates, including Qc^proteinase inhibitor fc^PI) and o^-raacroglobulin, and is eliminated finally as inactive enzyme-inhibitor complexes by the reticuloendothelial system. Using an enzyme-linked immunosorbent assay, we determined the plasma levels of E-fc^PI following major abdominal surgery, multiple trauma and pancreatogenic shock. Printed

with

the

permission

of

W.

de

Gruyter

Verlag

Proteinases in Inflammation and Tumor Invasion © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

Berlin-New

York

2 Whereas the operative trauma was followed by up to 3-fold increase of the E-ot]PI, postoperative septicemia was associated with a 10to 20-fold increase. The increase of E-O£JPI and a concomitant decrease of plasma factors, such as antithrombin III, clotting factor XIII and ft^-macroglobulin, were correlated. Multiple trauma caused a substantial increase of EoijPI up to 14 hours after accident. The released elastase seemed to correlate with the severity of injury, but assessing the relationship to consumption of plasma factors is complicated by concomitant transfusions. In acute pancreatitis, peaks of E-o£] PI coincided with a massive consumption of antithrombin III and o^-macroglobulin during shock.

Introduction

Severe injury or infection triggers the inflammatory response, including the activation of (a) such humoral systems as clotting, fibrinolysis, complement and kallikrein-kinin cascades, and (b) cellular systems, especially phagocytes, mast cells and lymphocytes, but also stress hormone producing cells. The humoral factors are often potent stimulators of the inflammatory cells, and conversely effectors from these cells may activate some humoral systems. Similarly, the repair mechanisms resulting from inflammation in their aggregate establish multiple relationships between the various parts of the organism (1).

Lysosomal proteinases We focus on the potential pathological role of lysosomal proteinases released extracellularly during inflammation. Phagocytes such as polymorphonuclear granulocytes and macrophages contain many lysosomes with a powerful hydrolytic and proteolytic potential (2). Normally, the lysosomal enzymes, along with oxidizing agents produced when phagocytosis is triggered, serve two main purposes (3): (a) intracellular protein catabolism including the degradation of intra- and extracellular endogenous substances, and (b) the degradation of phagocytized viruses and bacteria. In summary, lysosomal proteinases normally fulfill their physiological function inside the cell in the phagolysosomes, but under certain conditions, major escape of lysosomal factors from the cell occurs (3,19). Only small amounts leak out during normal

3 phagocytosis, higher amounts escape during frustrated phagocytosis, when the phagocyte is unable to take up a larger structural element such as a piece of vascular plasma membrane or cartilage. Disintegration of phagocytes caused by endogenous or exogenous endotoxins, possibly in combination with complement lysis, releases most or all lysosomal or phagolysosomal constituents (Figure 1 ).

L i b e r a t i o n

and

E f f e c t s

of

L y s o s o m a l

F a c t o r s

Endotoxins (endogenous J exogenous)

Antigen-antibody complexes Complement

| toxic peptides: blood pressure j, edema, heart toxic, clotting inh7

[

degradation/inactivation: membranes, connective tissue, plasma factors

activ. of system-specific proteases: thrombin, plasmin, C1-esterase, kal1ikrein

Figure 1. Liberation and effects of lysosomal factors during inflammation. Degradation products present in the phagolysosomes (e.g., toxic peptides) are liberated during cell stimulation or disintegration. Released lysosomal or phagolysosomal constituents degrade or inactivate native structural and humoral factors. System-specific proteinases activate the blood system cascades. In this way, activated proteinases may also degrade humoral factors, and therefore, toxic polypeptides may be generated as well.

Released extracellularly, lysosomal proteinases may enhance inflammation in two major ways (Figure 2): (a) selective proteolysis activates proenzymes, cofactors or both, and may generate biologically potent peptides such as kinins and anaphylatoxins. The

4

formed proteinases are eliminated by interaction with specific inhibitor proteins, as it is true for the specific consumption of factors participating in the clotting, fibrinolysis, complement and kallikrein-kinin cascades (called blood systems here); (b) unspecific proteolysis inactivates soluble factors such as antithrombin III, or digests structural elements (4-8). Degradation -induced unspecific consumption may produce toxic peptides as fibrin-fibrinogen degradation products which inhibit clotting (9).

Granulocytes

Macrophages

L i b e r a t i o n

1

S e l e c t i v e

Endothelial cells

of

proteolysis

P r o t e i

Unspecific

Mast cells

n

a s e s

II

proteolysis

• proenzyme/cofactor activation

•

inactivation by degradation

• biol. active peptides released

•

toxic

Specific

Unspecific consumption

consumption reaction

E + I

[EI J

AT I N .

peptides

produced reaction

• AT rainoctive

Figure 2. Reaction pathways caused by lysosomal proteinases if liberated from various body cells. E = enzyme; I = inhibitor; (EI) = enzyme-inhibitor complex; AT III = antithrombin III.

Clinical organ failure precipitated by severe inflammation or multiple injury affects primarily lungs, liver and kidneys. These organs are rich in endothelial cells, mast cells, fibroblasts, and macrophages, which contain many lysosomes. Above all, polymorphonuclear granulocytes may accumulate rapidly in the lungs during the inflammation. Hence, the relationships between the sequence of organ failures and the lysosomal content of different organs merits consideration. Among known lysosomal proteinases, the neutral proteinases of neutrophil polymorphonuclear granulocytes, elastase and cathepsin G are of special interest. They are stored in the lysosomes in fully active form, like the acid thiol and aspartate proteinases.

5 The most striking feature of both elastase and the chymotrypsin -like cathepsin G is non-specificity (3,10). Both enzymes degrade numerous humoral factors, including proteinase inhibitors (4,11), as well as structural elements, such as elastin (6) and collagen type III and IV (8), under physiological conditions (Figure 3). The digestive potential of lysosomes is demonstrated by the daily synthesis of over 1 g of neutral proteinases in man.

Substrates of PNN Granulocyte Neutral Proteinases P r o t e i n a s e * E l a s t a s e

B i o l o g i c a l

s u b s t r a t e s

- elastin, collagen I I I & IV, proteoglycans, FN* • clotting S fibrinolysis- factors • complement factors i immunoglobulins • proteinase inhibitors (AT l l l , a 2 PI, C1 INA; ITI) • transport proteins (transferrin, prealbumin)

C a t h e p s i n

G

• collagen II & I, proteoglycans, fibronectin - clotting 4 complement factors

C o l l a g e n a s e

- collagen I 4 II 4 I I I

* > 1 g daily turnover

+

fibronectin ( R E S

function)

Figure 3. Natural substrates of neutral proteinases from polymorphonuclear (PMN) granulocytes. AT III = antithrombin III; 2 P I = 2~Pl asm: '- n inhibitor; CI INA = Cl-inactivator; ITI = inter- -trypsin inhibitor.

Plasma proteinase inhibitors Functional aspects. Within the cell, lysosomal proteinases are kept under control first by their localization in membrane coated organelles, and second by proteinase inhibitors in the cytosol (12). Lysosomal proteinase escaping the cell face potent antagonists, such as the proteinase inhibitor proteins (13). «V^-Macroglobulin (O^M) effectively inhibits serine proteinases, as well as thiol (cysteine), aspartate and metallo proteinases. The high molecular weight of normally restricts its function to the vascular bed.

6 -Proteinase inhibitor (o^pi, formerly called e^-antitrypsin), the major antagonist of lysosomal neutrophil elastase, is present in high concentration in blood, but occurs also in interstitial fluid and mucous secretions. -Antichymotrypsin (5£-|AC), a rapidly responding acute phase reactant, reaching up to 6-fold concentration in response to inflammation, is a potent inhibitor of lysosomal neutrophil cathepsin G and mast cell chymase. The plasma concentrations of all other inhibitors are clearly lower. Still, the proteinase inhibitor proteins represent approximately 60% of the plasma proteins other than albumin and immunoglobulins . The interactions between proteinase inhibitors and their target enzymes in plasma are sketched in Figure 4. Excessive enzyme activation may be checked by three main inhibitors: antithrombin III (AT III) regulates clotting, »¿2-Plasm:i-n inhibitor (o£2PI) fibrinolysis, and Cl-inactivator (CI INA) both the classical complement pathway and the intrinsic coagulation cascade. This last function results from inhibition of plasma kallikrein Hageman factor or the 28 000 dalton Hageman factor fragment. The existence in plasma of complexes between V2-macroglobulin and kallikrein or plasmin suggests an involvement in the regulation of enzyme cascades in certain pathological conditions. However, the predominant role of ^¿2M s e e m s to be prevention of unspecific proteolysis by inhibiting all types of lysosomal proteinases including neutrophil elastase, cathepsin G, the thiol proteinases cathepsin B, H, L, the aspartate proteinase cathepsin D and metala lo enzymes such as collagenase. ^ 2M ^-so the most potent inhibitor of pancreatic trypsin which can activate enzyme cascades in blood and release kinins by selective proteolysis, or can unspecifically degrade various factors and structural elements while releasing toxic peptides. Despite this broad inhibitory n specificity, ^ 2 M °t a n acute phase reactant in man. O ^ PI strongly inhibits neutrophil elastase as well as pancreatic elastase, chymotrypsin, neutrophil cathepsin G and mast cell chymase (13).

Toxins (exogenous, endogenous) or

£

Cell

stimulation / disintegration

noxae

—c> P r o t e i n a s e

release

lysosomal elastase(s),

j

cathepsin Q*/B, H, L / 0

Unspecific degradation S i r inactivation ( 0 ; I • blood system factors • proteinase inhibitors • immunoglobulins • tissue etc. proteins phagocytes etc lymphocytes

Figure 4. Activation and consumption reactions caused by proteinases released during cell stimulation or disintegration: System-specific proteinases (e.g., thrombokinases and plasminogen activators) trigger activation of blood systems, whereby biologically highly active polypeptides are formed, e.g., SFMK (soluble fibrin monomer complexes) FDP (fibrinogen degradation products), kinins, and anaphylatoxins (C 3a and C 5a)(left part). Unspecific degradation or inactivation of plasma and tissue factors is caused by lysosomal proteinases and/or oxidizing agents (right part). In both cases, liberated polypeptides may stimulate suitable cellular systems. Finally, complex formation of activated or liberated enzymes (E) occurs with the proteinase inhibitors (I), e.g., (06,-proteinase inhibitor), V.2M ^¿¿-macroglobulin), V.1AC t>C| antichymotrypsin), AT III (antithrombin III), ®62PI (cig-plasmin inhibitor), and CI INA (Cl-inactivator). The enzyme-inhibitor complexes (EI) are eliminated by phagocytes of the reticulo-endothelial system (RES)(central part).

8 Inhibition of proteinases prevents formation of vasoactive or toxic peptides, such as kinins or anaphylatoxins, as much as stimulation by proteolytic products of cellular systems, such as thrombin-triggered platelet aggregation of anaphylatoxin induced chemotaxis of granulocytes. In essence, plasma proteinase inhibitors from equimolar complexes with their targets by which catalytic activity is irreversibly blocked. ^¿2M is a n exception to this rule, as it can bind two enzyme molecules, and the resulting complex can still cleave polypeptides below about 10 000 daltons (14,15). However, the enzyme-inhibitor complexes formed are rapidly cleared by the reticuloendothelial system (16).

Consumption of proteinase inhibitors by lysosomal factors. Reduction of the proteinase inhibiting potential by unspecific proteolysis is a striking pathological effect of lysosomal proteinases. Antithrombin III, for example, is rapidly inactivated by catalytic amounts of neutrophil elastase (4). The same is true for (b^-plasmin inhibitor and Cl-inactivator (11). Similarly, «¿.^-proteinase inhibitor is proteolytically inactivated by the lysosomal thiol proteinase cathepsin B, a metallo enzyme from macrophages, and by a bacterial elastase (13,17). Moreover, oxidation of the methionine residue in the enzyme-reactive site of greatly reduces the affinity to neutrophil elastase (18). Oxidizing agents as superoxide, hydroxyl radicals and hydrogen peroxide, are produced in large amounts in the phagolysosomes to facilitate intracellular protein breakdown. These substances may be released along with lysosomal enzymes in response to the same stimuli (3). Thus, injury or infection can induce consumption of proteinase inhibitor proteins in three ways: (a) complex formation with proteinases, (b) inactivation by proteolytic degradation, and (c) inactivation by oxidative denaturation. The last mechanism deserves special interest in connection with oxidative effects on «^-j PI on neutrophil elastase binding (Figure 5).

9

O x i d a t i o n and

Proteolysis

( M e t residuels) in a , P I oxidized to Met sulfoxide)

Figure 5. Affinity of native and oxidized 06|-proteinase inhibitor (a^PI) to polymorphonuclear (PMN) elastase (E) and pancreatic elastase (E), respectively. The oxidized PI reacts m u c h m o r e slowly (approximately 2000 times) with PMN elastase than native PI. In addition, the elastase complex with oxidized -|PI is dissociated by substrates with high aff inity to P M N elastase, thus again liberating the active enzyme. Oxidized PI does not react with pancreatic elastase.

Clinical studies Assay

of

liberated

neutrophil

elastase.

Liberated neutrophil elastase is found in blood primarily in the form of the e l a s t a s e - - p r o t e i n a s e inhibitor c o m p l e x (E-o^Pl). A small amount of neutrophil elastase may be bound to ^ - m a c r o g l o bulin, but the E-6i2M complex is much more rapidly eliminated from the circulation than the E-fc^Pl complex (t^f2 10' vs. 1 h). Consequently, assay of E-eigM complex in plasma requires extreme analytical sensitivity (16). This r e q u i r e m e n t may not apply in other body fluids, such as synovial fluid, where E-c^M complexes may be cleared slowly (20). We assay E-fc^Pl in clinical studies by an enzyme-linked immunoassay (Figure 6).

10

ab to + elastase

elastase« 1 PJ complex

— » ob-E/E-^PJ

+

abfoa^PJ labelled with alkaline phosphatase ( A P )

I ab-E/E-a-|PJ/ab«i PJ ( - A P ) substrate product photometer

00 405

Figure 6. Reaction scheme of the solid phase enzyme-linked immunoassay used for detecting the complex of polymorphonuclear (PMN) elastase (E) with ^¿1-proteinase inhibitor (C^PI) in biological samples. AP = alkaline phosphatase.

The assay is performed by (a) incubating standards (i.e., the complex produced in vitro) or unknowns for 1 h in polystyrene tubes coated with sheep antibodies to elastase, (b) washing, (c) incubating the tubes containing the fixed complex of elastase with cs^-j-proteinase inhibitor for 1 h with alkaline phosphatase labeled rabbit-antibodies to ^-proteinase inhibitor, (d) washing, and (e) determination of solid phase fixed alkaline phosphatase activity with p-nitrophenylphosphate as the substrate. The absorbance at 405 nm is measured and found to be linearily correlated with the concentration of the elastase-fc^ -proteinase inhibitor complex in the sample. The detection limit of the assay was 0.25 ng of complexed elastase in the test specimen. Normal plasma reacted linearly in this assay with sample volumes from 1 to 80 Recovery of the complex produced in vitro and added to different plasma specimens was excellent. Within-run variation (CV) with pooled plasma was 4 to 8%, between-run variation (CV) ranged from 3 to 8%. The normal range in citrated plasma was 98 + 26 ug/1 (mean + S.D., n = 43).

11

Elastase release induced by major surgery and septicemia. In a prospective study, plasma E-38.5° C; leucocytes >15.000 or ¿^5.000/mm 3 ; platelets 30% below the preoperative value; positive blood culture. Of these patients, fourteen survived (group B) while sixteen succumbed to septicemia (group C); eleven controls (group A) recovered without complications. Plasma levels of E-&£_.jPI are below 100 ng/ml in healthy individuals or preoperative patients without infection. The operation caused up to a 3-fold increase (Figure 7). Retrospectively, we noted that the preoperative mean value of group C was already elevated, possible due to increased concentrations in 6 patients infected before operation. Surgical removal of the infection focuses may have caused the slight mean decrease following operation. When septicemia was diagnosed clinically, E-a6jPI levels were increased manyfold in both groups B and C, with individual peaks as high as 2.500 ng/ml. With persisting septicemia, E-v-j PI remained high until death (group C), while recovery was accompanied by a decrease of E-V.-J PI to the normal range.

12

ng/ml

1000

Figure 7. Mean plasma levels of elastase-^-proteinase inhibitor complex (E-V,-|PI) in groups of patients subjected to major abdominal surgery: Group A patients (n = 11) being without postoperative infection; Group B patients (n = 14) surviving postoperative septicemia; Group C patients (n = 16) dying due to septicemia. The E-o^ PI levels are given as mean values (+ SEM) for the day before operation, the day after operation, as well as for the postoperative phase before sepsis, at onset of sepsis, and during septicemia. Last determinations were done on day of discharge (D) for Group A, on day of recovery (R) for Group B, and before death (D) for Group C. nr = normal range; ns = not significant; p = significance. Elastase release and plasma factor consumption in septicemia. Other indicators of inflammation were assayed in parallel with EO^i PI. Concentrations of antithrombin III, the most important inhibitor of the clotting cascade, were inversely related to E - ^ PI (Figure 8). Particularly at onset and during the course of septicemia, antithrombin III reached clinically critical concentrations posing the risk of hypercoagulopathy or disseminated intravascular coagulation. Similarly lowered concentrations were observed for fc^-macroglobulin and for coagulation enzyme factor XIII (Figure 9).

13

b e f o r e op.

a f t e r op.

before sepsis

• 001

< 0.0005

onset of s e p s i s

course of s e p s i s < 0 0005 < 0.0005 < 0.005

D

R

D

< 0.0005 . 0.0025

Figure 8. Mean plasma levels of the inhibitory activity of antithrombin III (AT III) in groups of patients subjected to major abdominal surgery. For details, see legend to Figure 7. nv = normal value; % of nv = percent of normal value (reference plasma = 1 00%).

Severe consumption of ^¿2-macroglobulin along with the carrier subunit of factor XIII (which is easily susceptible to degradation by lysosomal elastase) and the enzymatically active subunit (which is primarily consumed during clotting) evidences the imbalance among various blood enzyme cascades during septicemia. The concomitant elevation of E-fc^-) P I levels suggest the causal role of lysosomal proteinases released into circulation and there consuming proteinase inhibitor proteins.

14 Plasma levels, elevated ( 0 or decreased ( I ) : ( I I ) highly signif.

( I ) signif.

Parameter E - a , PI

(n) normal

Sepsis P r t f i n a l Survival complex

c

n

H

• I

a

II

II

n

xm

a

II

II

n

otj-Hacroglobulin

a

II

II

n

11 it

11 II

n-1

c

ft

tt

n

Antithrombin Factor

m

C - rtactivt protiin

o