Immunologically Active Peptides

137 Pages·1982·4.04 MB·English

by  Victor A. Najjar (auth.), V. A. Najjar (eds.)

Immunologically Active Peptides
Developments in Molecular and Cellular Biochemistry
Volume 2
V. A. NAJJAR, series editor
Already published:
l. V. A. Najjar, ed., The Biological Effects of Glutamic Acid and Its
Derivatives, 1981. ISBN 90-6193-841-4.
series ISBN 90-6193-898-8.
MARTINUS NIJHOFF/DR W. JUNK PUBLISHERS THE HAGUE/BOSTON/LONDON 1981
Immunologically Active Peptides
Edited by
v. A. NAJJAR
Reprinted from
Molecular and Cellular Biochemistry, Vol. 41, 1981
MARTIN US NIJHOFF/DR W. JUNK PUBLISHERS THE HAGUE/BOSTON/LONDON 1981
Distributors:
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Library of Congress Cataloging in Publication Data
Main entry under title:
Immunologically active peptides.
(Developments in molecular and cellular biochemistry; v. 2)
'Reprinted from Molecular and cellular biochemistry, vol. 41,1981.'
Includes index.
I. Peptides - Physiological effect - Addresses, essays, lectures.
2. Immunochemistry - Addresses, essays, lectures. 1. Najjar, V. A.
(Victor A.), 1914-. II. Molecular and cellular biochemistry. III. Series.
QP552. P4145 1981 616.07'9 81-20732
AACR2
ISBN- 13: 978-94-009-8032-7 e-ISBN-13: 978-94-009-8030-3
DOl: 10.1007/978-94-009-8030-3
Copyright © 1981 Martinus Nijhojjj Dr W. Junk Publishers, The Hague.
Softcover reprint of the hardcover 1st edition 1981
All rights reserved. No part of this publication may be reproduced, stored in a retrieval
system, or transmitted in any form or by any means, mechanical, photocopying,
recording, or olherwise, wilhouc Ihe prior wriuen permi,sion of the publishers,
Martinus Nijhoff/ Dr W. Junk Publishers, P.o. Box 566,2501 CN The Hague, The Netherlands.
Contents
Introduction by Victor A. Najjar ............................................... .
Victor A. Najjar, Danuta Konopinska, Mansa K. Chaudhuri, Donald E. Schmidt and Lisa Linehan:
Tuftsin, a natural activator of phagocytic functions including tumoricidal activity ... . . . . . . . . 3
Kenji Nishioka, George F. Badcock, loseph H. Phillips and R. Dirk Noyes:
Antitumor effect of tuftsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Henry J. Showell, William M. Mackin, Elmer L. Becker, Nakesa Muthukumarasway, Alan R. Day and
Richard Freer:
N a-formyl-norleucyl-phenylalanyl chloromethylketone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Edgar Lederer, Arlette Adam, lean-Fran«;ois Petit and Pierre Lefrancier:
Muramyl peptides ........................................................ 27
Shu-Kuang Hu, Teresa L. K. Low and Allan L. Goldstein:
Modulation of terminal deoxynl!cleotidyl transferase activity by thymosin ................ 49
Tony E. Hugli, Craig Gerard, Marleen Kawahara, Maurice E. Scheetz II, Russel Barton, Stephen
Briggs and Gary Koppel:
Isolation of three separate anaphylatoxins from complement-activated human serum. . . . . . . . . 59
lean-Marie Pleau, Mireille Dardenne and Jean-Fran«;ois Bach:
The serum thymic factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Mati Fridkin and Philip Gottlieb:
Tuftsin Thr-Lys-Pro-Arg. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Ignacy Z. Siemion and Danuta Konopinska:
Tuftsin analogs and their biological activity ...................................... 99
M. Bruley-Rosset, I. Florentin and G. Mathe:
Macrophage activation by tuftsin and muramyl-dipeptide ............................ 113
1 ean- Louis Touraine:
Induction of T lymphocyte differentiation by thymic factors. . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Jean Martinez and Fran«;ois Winternitz:
Bacterial activity of tuftsin .................................................. 123
Introduction to Developments in molecular and cellular biochemistry
Molecular and Cellular Biochemistry is an international journal that covers a wide range of biophysical,
biochemical and cellular research. This type of coverage is intended to acquaint the reader with the several
parameters of biological research that are relevant to various fields of interest. Unlike highly specialized
journals, it does not bring into focus a particular field of investigation on a monthly basis. Accordingly, it has
been decided to supplement its present wide scope by periodic presentations of a restricted area of research in
the form of book-length volumes. These volumes will also be published in hardcover as a book series
entitled, Devr?!opments in Molecular and Cellular Biochemistry. Each volume will focus on an active topic of
interest which will be covered in depth. It will encompass a series of contributions that deal exclusively with
one single well-defined subject.
The present volume, Immunologically Active Peptides, is the second one in the book series. The first one
deals extensively with The Biological Effects oj' Glutamatic Acid and Its Derivatives.
It is the editor's hope that M oletular and Cellular Biochemistry will fulfill its intended role of service to the
international community of biological scientists.
Immunologically active peptides
No area of science, in this decade, has witnessed a greater expansion than the science of immunology, both
cellular and molecular. The mere classification of the various types of lymphocytes, both immunochemically
and biologically, continues unabated. The phagocytic cells have bounced back from relative obscurity to
well-deserved prominence. The macrophage is a world of its own. The so-called 'scavenger', a grave insult to a
noble cell, has now become the hub of immunology as it sits at the convergence of cellular and numoral
immunology. It is at once a sequesterer and killer of invading bacteria and syngeneic cancerous cells, as well
as, the processor of the antigen for anti-body response. Such a cell, one would think, is entitled, in its own
right, to a natural activator that regulates its function. Various factors have one or more qualifications for this
role. Several articles in this volume bear on this point. Little need be said for the various lymphokines,
thymosin, thymic serum factor, etc., as these polypeptides have already taken a well-earned place in the realm
of immunology. Several articles deal with this aspect of biology and function.
Victor A. Najjar,
Editor in chief
Tuftsin, a natural activator of phagocytic functions including tumoricidal activity
Victor A. Najjar, Danuta Konopinska, Manas K. Chaudhuri, Donald E. Schmidt* and Lisa Linehan
Division of Protein Chemistry, Tufls University School of Medicine, Boston, M A 02111, * Rainin
Instruments Co., Inc., Woburn, MA 01801, U.S.A.
Summary
Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three
phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-Lys-
Pro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue
aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in
prolonging the life of syngeneic mice injected with Ll210 leukemia cells. Its effect in our laboratory, was
considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high
performance liquid chromatography is described using 0.75% trifluoroacetic acid in water.
The tuftsin sequence Thr-Lys-Pro-Arg is present in P 12 protein of Rausher murine leukemia virus. A close
analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second
close analog Thr-Arg-Pro-Arg forms the penultimate carboxy terminal of a pancreatic polypeptide found in
human and several animals.
Introduction A good deal of novel and unique observations
preceded the identification of tuftsin (6, 7). These
Over four dozen compounds have so far been findings gave rise to the conviction that it is a true
reported to activate phagocytic cells; principally the biological entity with the assigned function of
macrophage. The mechanism of activation is a activating phagocytic cells: (a) Tuftsin is a part of a
highly complex process that involves the stimula- specific carrier yG, cytophilic y-globulin, leukoki-
tion of several functions of the phagocyte. These are nin (8). (b) The carrier leukokinin circulates through
primarily phagocytosis, kinesis, immunogenic the spleen where it is nicked by an enzyme, tuftsin-
activity hexose monophosphate shunt, bactericidal endocarboxypeptidase, between residues 292 and
activity and most importantly tumoricidal activity. 293 (Arg-Glu) of the heavy chain. This nicked
It is hardly possible that nature would devise molecule, leukokinin-S (2), then binds to specific
such a highly elaborate and complex system only to receptors on the outer membrane of the phagocyte
be triggered by compounds external to the body where the enzyme leukokininase cleaves the tetra-
such as bacteria and bacterial products. Nature peptide off the parent molecule between residues
would do better. It would devise a natural intrinsic 288 and 289 of the heavy chain (Lys-Thr) to release
compound(s) primarily designed for the sole pur- active tuftsin (2). Thus the phagocytic cell plays a
pose of regulating the physiological functions of unique role in releasing its own activator tuftsin. (c)
phagocytic cells. Among these is a compound Finally, a mutation affecting tuftsin has been
which was discovered a few years ago in human identified. It results in a specific human syndrome
serum is tuftsin (1-5) a tetrapeptide, Thr-Lys-Pro- with increased frequency of severe infections.
Arg. Seventeen such mutants have been identified in the
Molecular and Cellular Biochemistry 41, 3-12 (1981). 0300 -8177 /81/0041 0003/$02.00.
© 1981, Martinus Nijhoff/ Dr W. Junk Publishers,
4
United States of America (9-11) and two in Japan preserved its fundamental structure. It is present in
(12). (d) Another type of tuftsin deficiency is some analogous form in all animals studied. It has
encountered after removal of the spleen (8-11, 13, been isolated in pure form from man and dog (2). In
14). Here the tetrapeptide fails to be nicked at Arg- the latter, the carboxy-terminal residue, arginine,
Glu and consequently remains bound to leukokinin of human tuftsin is replaced by lysine. The resulting
and as such is inactive. This type of intact leukoki- analog exhibits comparable biological activity. In
nin still binds as tightly to its cell receptors as the rabbit, immunochemical studies indicate that
leukokinin-S which is already nicked at Arg-Glu the second, third and fourth residues from the
(Table I). The table shows that in low ionic strength amino terminal are the same as in man (13, 14).
isotonic buffers, the bindingto specific cell receptors Furthermore, in mice 'Y-G I, MOPC-21 a glutamine
is equally strong for leukokinin-S and for unnicked, residue replaces lysine, the second residue of tufts in
intact and inactive leukokinin. This indicates that (16).
bound leukokinin of either type displays tight and
continuous association with the cell surface. Con- The activation of the phagocytes
sequently, the receptor site is distinct and separate
from the site of leukokininase activity. The functional expression of tuftsin is exerted in
The foregoing findings which are detailed in three phases:
several reviews (2, 3, 15) support the concept that Phase J is expressed in rapid stimulation of the
tufts in is a real biological entity with a specific phagocytic and pinocytic activity ofthe neutrophilic
defensive function exerted on phagocytic cells, granulocyte and the macrophage. There is also a
which are the only cells that possess specific tuftsin concomitant stimulation of the hexose monophos-
receptors (13). The importance of an activator like phate pathway.
tuftsin is reflected in the fact that nature has Phase JJ is a more gradual process that extends
Table I. Serum was obtained from dog #25 a week before splenectomy (leukokinin-S) and
three months after splenectomy. Leukokinin was prepared from serum -y-globulin by
fractionation (Fraction IV) on phosphocellulose columns (32). Phagocytosis assay (7-9)
was carried out with dog buffy coat cells in isotonic buffered sucrose using Ieukokinin
prepared from serum obtained before splenectomy, experiments 1-4, and after sple-
nectomy, experiments 5-S. Assays were further carried out with a mixture of the two types
of leukokinin but with a definite order of addition. In the first addition, cells were allowed to
bind added leukokinin for 5 min after which the second addition was made along with target
particles Staphylococcus aureus. Note that phagocytosis stimulation was determined by the
type of leukokinin used in the first addition showing tight binding to membrane
receptors. Phagocytic index represents the number of cells containing bacteria! 100
phagocytic cells counted usually 400- 500 cells were counted.
Experiment Order of addition of leukokinin prepared Leukokinin PhagoCJ'/osis
number from serum obtained before (Ieukokinin-S) each addition index
or after splenectomy (Dog #25) (fig) (%)
I st addition 2nd addition
I. Before splenectomy None 0 IS
2. Before splenectomy None 50 35
3. Before splenectomy None 100 42
4. Before splenectomy None 200 41
5. After splenectomy None 0 16
6. After splenectomy None 50 IS
7. After splenectomy None 100 21
S. After splenectomy None 200 19
9. Before splenectomy After splenectomy 100 40
10. Before splenectomy After splenectomy 200 45
II. After splenectomy Before splenectomy 100 22
12. After splenectomy Before splenectomy 200 19
5
for a period of a few hours during which there is a four different species of bacteria were killed
strong stimulation of motility. At the same time the more rapidly after ingestion by tuftsin activated
engulfed particle or antigen is degraded or pro- macrophages than by control cells not exposed
cessed for presentation to the antibody forming to tuftsin. This bactericidal stimulation paral-
cell. leled the increased blood clearing of these
Phase III is a much slower process than the first bacteria following tuftsin injection of 10-20
two and requires several days. This phase or in mgj kilo of body weight.
combination with other phases, in one way or
another, works towards the maturation and activa- Phase II:
tion of the large spreading phagocytic cell to
augment its bactericidal activity and to render it (a) Stimulation ofmotility ufhlood J;ranulocytes.
highly cytocidal towards syngeneic tumor cells. In contrast to phagocytic stimulation, the
Details of the three phases follow: increase rate of motility requires considerably
higher concentrations of tufts in, approximately
Phase I: fiftyfold. At 5 MM there was definite increase in
motility, approximately a third of maximal.
(a) Increased phagocytic activity. Increased pha- However at 25 MM, maximum stimulation was
gocytic and pinocytic activity is an immediate attained. Thus at a concentration that yields a
and highly specific effect of tuftsin. It can be half maximal phagocytic effect 0.10 MM, no
measured within a few minutes after the expo- stimulation of motility could be discerned. This
sure of blood granulocytes or tissue macro- is of particular importance since the concentra-
phages to low concentrations of tuftsin. The tion available to the blood neutrophil in human
concentration that results in half maximal serum is approximately 0.3 MM (14). Conse-
phagocytic activity (Km) of either phagocytic quently, it is difficult to assign a definitive
cell is about \ 00 17M (0.05 Mgj ml) (3, \3-\5). biological role for tuftsin stimulation of motility
Tuftsin is incapable of stimulating pinocytosis in view of the high concentration required
of 3T ,2 , 3Tfi and Ll210 cells (15) as well as unless the effective concentration is much
HeLa cells (Constantopoulos, A. and Najjar, greater. This could well be the case inasmuch as
V. A., unpublished). tuftsin carrier leukokinin is bound to cell
receptors in saturating amounts at normal
(b) Increased stimulation of the hexosemono- serum levels (Table I). Furthermore, tuftsin is
phosphate shunt. It has been known for some- released on site.
time that phagocytosis is accompanied by
stimulation of the hexose mono phosphate (b) Stimulation of the immunogenic function of
shunt with its attendant increase in hydrogen the macrophaJ;e. This phase occurs after pro-
peroxide, superoxide, hydroxyl radicals and longed exposure of the macrophage to the
singlet oxygen (17). The high level of these antigen during which a T helper c~lI, in the blast
active compounds is assumed to result in the stage, would interact with the antigen process-
halogenation of the ingested particle or a direct ing macrophage. The presence of tuftsin during
effect of these compounds on the target particle this time stimulates considerably the little
with its consequent destruction and elimina- understood phenomenon of processing of the
tion, be it a bacterium, a dead cell or an antigen for maximal immunogenic stimula-
abnormal cell (IS, \8). tion. Mouse peritoneal macrophages were
Stimulation ofthe metabolic shunt by tuftsin, exposed to thymus dependent antigen with and
as measured by the reduction of the nitrotetra- without tuftsin (21). After a few hours of
zolium dye, was shown by Spirer et al. . (\9). It incubation, the washed macrophages were then
follows that under these conditions there would incubated with nonadherent splenic cells. These
be expected an increased rate of killing of were later irradiated and injected into syngeneic
ingested bacteria. This was indeed the case. mice. After several days, the lymphocytes of the
Martinez et al .. (20) showed conclusively that draining Iymphnodes were shown to take up
6
[3H]-thymidine over seven times the uptake of (b) Stimulation olthe tumoristaticand tumoricidal
similar lymphocytes obtained from the corre- activity of the macrophage by tuftsin and
sponding controls where no tuftsin was used tuflsinyltuftsin. A good deal of work has been
during antigen processing. Maximal stimula- done to establish the fact that mononuclear
tion was obtained at tuftsin concentration of cells including tissue macrophages, blood
approximately 5 MM. Concentrations on either monocytes and granulocytes can exert antine-
side of the optimum result in a diminished oplastic properties (24, 25). The question natu-
effect (21). It is our opinion that this regulatory rally arises as to whether tuftsin can augment
pattern is modulated by the relative concentra- such properties inasmuch as it is the natural
tions of tufts in and the tripeptide Lys-Pro-Arg, activator of these cells (15).
a product of the action of an aminopeptidase Florentin et aI., (22) and Nishioka (23)
on tuftsin and which is a good inhibitor of showed that after tuftsin-treatment, mice de-
tuftsin stimulation of phagocytosis (13). veloped a higher level of cytostatic and
The demonstration at the Weizmann Insti- cytocidal activity of their macrophages. Fur-
tute of the immunogenic stimulation by tuftsin thermore, there was also an enhancement of
which is a distinct part of the repertoir of the antibody-dependent cell-mediated cytotoxicity
macrophage may be translated into a direct (22). This activation of the macrophage by
augmentation of antibody production by anti- tuftsin resulted in an in vivo augmentation of
body forming cells. This indeed proved to be mice survival following injections with L12 \0
the case as demonstrated clearly by Florentin cells and suppression of the growth of injected
et al., (22). They showed that tuftsin injected melanoma cells (23).
into mice at a precise number of days before Another successful demonstration of the
antigen administration, increased antibody antitumor activity of tuftsin in vivo has recently
response significantly to T depenqent and been reported by Catane et al., (26). They used
independent antigens as well as augmented the 3-methyl-cholanthrene induced transplantable
antibody-dependent cell mediated cytotoxicity. fibrosarcoma injected intra peritoneally, in C3H
mice. This was uniformally lethal in control
Phase Ill: animals with a mean survival time of 21 days.
However, with the intraperitoneal injection of
(a) Stimulation o.f maturation and differentiation 10-500 Mg/ kg of body weight, the mean survival
of the macrophage. There have been of late, time was 39 days (p < 0.001) and 20% of the
several instances where the effect of tuftsin mice survived beyond 80 days.
administration to an animal requires several Our laboratory has been similarly engaged in
days to reach full fruition. Thus the experi- a parallel effort for the study of the effect of
ments reported by Florentin et al., (22) clearly tuftsin on the survival of L12 \0 infected mice.
show that, following tuftsin administration to We obtained increased survival of treated mice
mice, several days must elapse before antigen but not of sufficient magnitude. It was our
challenge at which time the recipient animal is opinion that with the most active preparation
fully prepared to mount a maximal immune of tuftsin, the formation of the tripeptide Lys-
response. For T dependent antigens, seven days Pro-Arg, a natural inhibitor of tuftsin (13),
must elapse before antigen injection and only might explain the limited effect obtained. There
1-3 days for T independent antigens, while are active cytosol and membrane aminopepti-
antibody-dependent cell mediated cytotoxicity dases that would yield this inhibitor peptide
require seven days for its fullest expression. from tuftsin ( 15). Consequently, we synthesized
These findings parallel the results obtained by several analogs in order to evaluate this anti-
Nishioka (23) showing that 5-7 days after the tumor activity with the primary purpose of
intraperitoneal injections of tuftsin in mice, minimizing or eliminating the formation or the
there was a considerable augmentation of the accumulation of Lys-Pro-Arg. Among these
number of mature spreading intraperitoneal are Ala-Lys-tuftsin, acetyl-Ala-Lys-tuftsin,
macrophages. Ala-Lys-tuftsin-Glu-Ala-Ala-Ala, [Glu2]-tuft