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COMPLEMENT ACTIVE MATERIALS _ ~
FROM LARVAL TAENIIDS

Dissertation for‘vthe Degree of Ph. ‘D. I
MICHIGANSTATE UNIVERSITY ' -
‘ BRUCE'HAMMERBERG" '
1977

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COMPLEMENT ACTIVE MATERIALS
FROM LARVAL TAENI IDS

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ABSTRACT

COMPLEMENT ACTIVE MATERIALS
FROM LARVAL TAENIIDS

BY

Bruce Hammerberg

Non-specific complement-fixing activity was found in bladder
fluid and cyst fluid of larval stage taeniid cestodes. Larval
T. taeniaefbrmis was shown to produce complement active material
during in vitro maintenance and to shed this substance when incubated
in a Tris—HCl buffer containing EDTA. The biological activity
of these materials was characterized and physico-chemical analyses
of larval extracts was conducted.

The results of the characterization of larval material
interactions with complement demonstrated the depletion of hemolytic
complement in normal sera of several species, the generation of
anaphylatoxin-like activity in vitro, the conversion of C3, and the
production of vascular permeability changes in viva. Complement
fixation appeared to be initiated non-immunologically via both the
alternative and classical pathways. Parasite factors given
intravenously to rats led to profound depression of circulating
complement levels and repeated dosing over a 3 week period did not
cause the rats to become refractory to this effect. Circulating

levels of hemolytic complement were not altered in infected rats.

Bruce Hammerberg
Complement-dependent inflammatory responses in the skin of rats
were inhibited in animals given intravenous doses of parasite factors.

Physico-chemical analysis of materials extracted from T.
taeniaeformis larvae was accomplished by using enzymatic digestion,
gel-filtration, anion exchange chromatography, ultracentrifugation
and gel electrophoresis. The results of these analyses provided
evidence of an association between antihemolytic activity and
macromolecules containing carbohydrate, protein, sulphate and
hexosamine, heterogenous in net charge but resistant to proteolysis
and beta elimination reactions, and free of sialic and uronic acids.
The anion exchange chromatography fraction with the greatest activity
was eluted from diethylaminoethyl cellulose with 0.3 M sodium
chloride in 0.01 M Tris-HCl pH 8.0 and when parasites were incubated
with a source of 35804", radioactivity was incorporated into
molecules eluting under identical conditions. Active chromatographic
fractions precipitated with protamine sulphate suggesting that they
were polyanionic in nature. There was some evidence of dissociation
of the active substance on polyacrylamide gels where fast-moving
sulphate containing bands were observed which no longer reacted
with protamine or complement.

The results are consistent with the possibility that the
active substance is a polysulfated proteoglycan. These types of
molecules have been detected on the surface of a variety of infectious
organisms, and are known to interact with complement non-immunologically.
The location of these molecules at host-parasite interfaces may

be significant in parasite evasion of immune rejection.

COMPLEMENT ACTIVE MATERIALS

FROM LARVAL TAENIIDS

BY

Bruce Hammerberg

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1977

A

Dedicated to my wife JoAnn

ii

ACKNOWLEDGEMENTS

I am greatly indebted to Dr. Jeffrey F. Williams for sound
and stimulating academic guidance, thorough and accurate evaluations
of data resulting from my thesis research, and invaluable assistance
in composing manuscripts for publication. His efforts in obtaining
financial support for me from the NIH have made it possible for
me to complete my work in the shortest period of time possible.

Most important of all and sincerely felt is the warm and relaxed
personal communication which he encourages.

The advice and encouragement of my guidance committee,

Drs. Harold Miller, Richard Patrick, Wayne Smith, and Donald Twohy
are gratefully acknowledged.

The camaraderie of those working with me in this laboratory
during the last three years has made this experience very enjoyable.
For this companionship, I thank all the members of Dr. Williams'

laboratory.

iii

TABLE OF CONTENTS

LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . .

Immune Avoidance Mechanisms. . . . . . . . . . . . .
Antigenic variation . . . . . . . . . . . . .
Masking effects . . . . . . . . . . . . . . .
Immunosuppression . . . . . . . . . . . . . .

Role of Complement in Specific and Non-specific

Immunity . . . . . . . . . . . . . . . . . . . . . .
Complement fixation . . . . . . . . . . . . .
The involvement of complement in resistance
to parasites. . . . . . . . . . . . . . . . .

Mechanisms of Resistance to Lysis. . . . . . . . . .
Tissue location . . . . . . . . . . . . . . .
Surface coat characteristics in bacteria. . .
Surface polyanions and their potential role
in resistance in lysis. . . . . . . . . . . .
Parasite surface characteristics and evidence
for the presence of polyanions. . . . . . . .

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . .

ARTICLE 1 - ACTIVATION OF COMPLEMENT BY HYDATID CYST FLUID
OF ECHINOCOCCUS GRANULOSUS. . . . . . . . . . .

ARTICLE 2 - INTERACTION BETWEEN TAENIA TAENIAEFORMIS AND
mE COW MNT SYS TEM . O O O O O C O O I C O 0

ARTICLE 3 - PHYSICO-CHEMICAL CHARACTERIZATION OF COMPLEMENT-
INTERACTING FACTORS FROM TAENIA TAENIAEFORMIS .

iv

Page

H

0101.50.»

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23
23
24
25
28

33

45

64

95

LIST OF TABLES

Table Page
ARTICLE 2
1 Percent depression of total hemolytic complement
levels . . . . . . . . . . . . . . . . . . . . . . . . 74
2 Effect of differential cation chelation on C3

consumption by products of T. taeniaefbrmis. . . . . . 76

LIST OF FIGURES
Figure Page
ARTICLE 1

l Ouchterlony analysis of rat hydatid fluid (HF)
before and after absorption of host serum

proteins (HF Abs). . . . . . . . . . . . . . . . . . . 52
2 Immunoelectrophoretic analysis of human serum

following incubation with either hydatid fluid

or zymo s an O O O O O O O O O O O O O O O O O O O O O O S 2
3 Smooth muscle contraction in response to sequential

Stimulation. . O O O O O O O O O C O C O O C C O C O C 55

4 Response to intradermal inoculation of 0.05 ml
RHCF-l (rat hydatid fluid fraction) in normal
human subjects . . . . . . . . . . . . . . . . . . . . 58
ARTICLE 2
1 Liver of rat infected with cystic larvae of

Taenia taeniaeformis and larvae dissected from
within the host capsule. . . . . . . . . . . . . . . . 70

2 Immunoelectrophoretic analysis of human serum
following incubation with either CYF or zymosan. . . . 78

3 Smooth muscle contraction in response to
sequential stimulation . . . . . . . . . . . . . . . . 80

4 Serial determinations of serum complement (CHSO)
levels of rats dosed with 1000 eggs of Taenia
taeniaefbrmis at 28 days of age. . . . . . . . . . . . 84
ARTICLE 3

1 Effect of acid hydrolysis of T.E. on inhibitory
activity in a complement-mediated hemolytic assay. . . 104

2 Protein and neutral sugar elution profiles of IVP

and T.E. from a Sepharose 6-B column equilibrated
with 0.1 M Tris-HCl buffer, pH 8.0 . . . . . . . . . . 106

vi

Figure Page

3 Protein and neutral sugar elution profile of T.E.
from a Sephadex G-200 column equilibrated with
2 M TaCl in 0.1 M Tris-HCl, pH 8.0 . . . . . . . . . . 108

4 Protein and neutral sugar elution profile of pepsin-
digested and pepsin-digested plus NaOH-treated T.E.
from a Sepharose 6-8 column equilibrated with 0.1 M
Tris-HCl buffer, pH 8.0. . . . . . . . . . . . . . . . 110

5 Comparison of the protein and neutral sugar elution
profiles of T.E. before and after digestion with
pepsin . . . . . . . . . . . . . . . . . . . . . . . . 113

6 Relationship of protein and neutral sugar to cpm

from 35804" and complement-inhibitory activity

in fractions of IVP from DEAE cellulose. . . . . . . . 115
7 Precipitation bands formed between protamine

sulfate and T.E. fractions . . . . . . . . . . . . . . 118
8 Polyacrylamide disc gel electrophoresis band
pattern typical of T.E. or T.E. after alkaline

hydrolysis or pepsin digestion . . . . . . . . . . . . 120

9 Polyacrylamide gel electrophoresis of IVP
labelled with 35504". . . . . . . . . . . . . . . . . 122

vii

LITERATURE REVIEW

Justification for research in experimental taeniiasis is
two-fold. Firstly, the experimental cysticercoses provide a
convenient model for the cyclozoonotic cestode infections which
are of major public health and economic importance in many parts
of the world. There is a need for an improved understanding of
the immune response in these infections, both from the viewpoint
of its potential exploitation in a preventive way and in terms
of the development of immunodiagnostic procedures. Secondly,
the larval cestodes are able to survive in tissues for prolonged
periods and any clarification of the mechanisms which contribute
to this phenomenon are relevant to infectious disease processes
as a whole.

In recent years there have been ample reviews of the global
impact of cysticercosis and hydatidosis (Schenone, 1974; Abdussalam,
1974). These reviews of the current situation reflect the increas-
ing importance of these infections, and the less than impressive
progress made in control programs, and emphasize the need for
immunological means of inducing resistance. There is ample
evidence from field studies of the occurrence of an acquired
immunity to taeniiasis (Gemmell and McNamara, 1972), and the
evidence derived from studies of immune mechanisms in the laboratory

animal models have been extensively discussed by Leid (1973).

Musoke (1975) and Hustead (1976). These reviews point up the paradox
of coexistence of a high degree of acquired immunity in animals
which harbor persistent infections throughout their lives.

The issue of prolonged survival of parasites in immune hosts
and the question of how tissue-dwelling organisms interact with
specific and non-specific elements of the immune response have not
been reviewed from the perspective of the common problems faced
by invasive protozoa, helminths and bacteria. This dissertation
deals with substances which have demonstrable interactions with
the complement and coagulation cascades and therefore may have
a significant role in determining the outcome of host-parasite
confrontation. For that reason an attempt is made in the lit-
erature review to describe current hypotheses on prolonged survival
of infectious organisms. The demonstratable effects of polyanions
are discussed and their potential role in the relationship between
cells and resistance to immune attack mechanisms is outlined. A
comparable role is proposed for molecules on the surface of

parasites.

3

I. IMMUNE AVOIDANCE MECHANISMS

Infectious agents ranging from viruses to metazoan organisms
multiply and/or undergo obligatory morphologic development in
immunologically competent hosts. In some instances acute disease
syndromes occur in which the host immune system is effective in
eliminating the organisms but not before significant reproductive
or morphological changes have occurred which facilitate the survival
of the parasite in a new environment, or permit its transmission
to a non-immune host or vector. Propagation of many eukaryotic
and protozoan parasites is therefore linked to the delay in the
primary immune response and to the ability of the parasite to
evolve or divide rapidly. 0n the other hand, most multicellular
infectious organisms, and some protozoan and bacterial organisms,
do not develop or divide with sufficient quickness to leave the
host before the onset of the primary immune response. In these
host-parasite relationships avoidance mechanisms have evolved
which result in the evasion of specific immune recognition and/or
rejection systems. This characteristic is a hallmark of

.
helminthiases of medical and veterinary importance and the variety
of evasive mechanisms which have been described will be reviewed
here. There is a wealth of information on evasive mechanisms in
the broader context of chronic infectious disease which is very
relevant to the study of helminthiasis, and that also will be
reviewed.

Currently, much more is known about bacterial products which
contribute to avoidance of or interference with the host immune

system, than about helminthic or protozoan factors. This is

4
undoubtedly attributable, in part, to the advantage which bacteriolog-
ists have in being able to select and culture specific organisms
and strains. In a recent review of bacterial factors which affect
host rejection mechanisms Glynn (1972) described substances in the
category of bacterial "impedins" which; inhibit local inflammatory
responses (Hill, 1968), non-specifically aggregate immunoglobulins
and depress phagocytosis (Dossett et a1, l969),provide surface
barriers to complement-mediated lysis (Glynn and Howard, 1970),
and non-specifically suppress host cell-mediated immunity (Bullock,
1968). There is an abundance of information on bacterial lipopoly-
saccharides and their function in immune evasion (Glynn, 1972).
Comparable data are presently unavailable for non-bacterial parasite
avoidance mechanisms, but the current state of knowledge was reviewed
in a Ciba Foundation Symposium (1974) and some recent developments

have been highlighted by Ogilvie and Wilson (1976).

a. Antigenic variation

 

Antigenic variation which occurs in protozoan infections
of mammals has been investigated by numerous researchers and this
work was reviewed recently for Plasmodium (Brown, 1974), Trypanosoma
(Vickerman, 1974), and Babesia (Greenwood, 1974). Those antigenic
changes occurring on the parasite surface are believed to be elicited
by specific antibody binding (Seed, 1977). They may be induced
phenotypically when antibody-binding leads to capping and elimin-
ation of the former antigens without parasite lysis. Alternatively,
antibody binding and subsequent lysis may result in the emergence
of genetically distinct parasites which go undetected until the

new antigen is recognized by the host (Vickerman, 1974). In the

African trypanosomes the variant antigens are located in the surface
coat, which is distinct from the cell membrane. When shed in vivo
from live trypanosomes, these antigens are found in the circulating
blood of infected animals and are also known to be released from

the parasite at a.pH lower than 7.3 (Allsopp et a1, 1971). Removal
of variant antigens from T. brucei by tryptic digestion reveals a
carbohydrate layer, demonstrable by periodic acid-silver staining,

external to the plasma membrane (Wright and Hales, 1970).

b. Masking effects

 

Another mechanism of immune avoidance which involves alteration
of surface antigens has been postulated, based on evidence that
schistosomes are able to selectively acquire or produce host antigens
(Smithers et a1, 1969). Clegg (1974) showed with Schistosoma
mansoni that invading schistosomula lacked host red blood cell
antigens three hours after penetration and were susceptible to immune
attack; whereas, four days after penetration schistosomula had acquired
red blood cell antigens and were able to avoid antibodies from immune
serum. Whether or not the acquisition of host antigens is responsible
for the prolonged survival of mature schistosomes or is merely
coincidental has not yet been established (Dean, 1977). Because
the schistosome surface is the site of a dynamic antigen flux and
also is the site of immune attack it clearly plays an important

role in avoidance of immune rejection (Ogilvie and Wilson, 1976).

c. Immunosuppression

 

Many parasites which undergo antigenic variation or show host
antigen uptake are known to shed soluble antigens into the host

circulation. Other protozoa and helminths such as Plasmodium

(Wilson et a1, 1969), Babesia (Zuckerman and Ristic, 1968), Trypanosoma
(Williamson and Brown, 1964), Haemonchus (Stumberg, 1933), and
Trichinella Bozicevich and Detre, 1940) are known to release cir—
culating antigens. The observation that host immune systems may

be depressed has led to speculation that these antigens may function

as blocking agents via antigen-specific immunosuppression of lympho-
cytes or by non-specific saturation of the reticulo-endothelial

system (Wilson, 1974).

More detailed mechanisms of soluble antigen action on immune
systems have been proposed but evidence that these function in vivo
to the advantage of the parasite has yet to be developed. For example
there may be antigenic competition at the macrophage-level inhibiting
phagocytosis or blocking cooperation between T and B lymphocytes;
there could be stimulation of suppressor T cells; binding and
subsequent neutralization of high affinity antibodies may occur;
and soluble circulating antigen-antibody complex formation has been
proposed to block killer cell action against the parasite (Ciba
Foundation Symposium, 1974).

Whatever the mechanism, instances of immunosuppression in
parasitic infection are now widely recognized. Circumstantial
evidence from the comparison of immune competence of normal humans
with parasitized subjects indicates a nonspecific depression of
antibody response to Clostridium tetani and Salmonella typhi vaccines
during Plasmodium falciparum and Trypanosoma gambiense infections.
Also, T. gambiense infection inhibits the response of patients
to skin tests for PPD or Candida antigen, and reduces susceptibility
to skin sensitization with dinitrochlorobenzene (Greenwood, 1974;

Wedderburn, 1974). On the whole these results indicate suppression

of humoral immunity during malarial infections, whereas trypanosomiasis
may lead to suppression of both humoral and cell-mediated immunity.

In malarial infections in mice (P. berghei yoelii), acute
stage immunosuppression of the IgG response may be due to an effect
of macrophage functioning, such as antigen presentation to T or B
cells, but is not apparently attributable to failure of antigen
uptake by macrophages (Greenwood, 1974). In acute infections with
Trypanosoma brucei in mice (Terry et a1, 1973; Freeman et a1, 1974)
the IgG response to sheep red blood cells is drastically reduced and
this effect is completely reversible upon elimination of the parasite.
In chronic infections both IgM and IgG responses are greatly depressed
and there is no recovery of normal responsiveness after clearance
of the parasites.

Generalized immunosuppression has been demonstrated by enhanced
tumor growth in rats infected with Nippostronglyus brasiliensis
(Keller and Jones, 1971), and in hamsters infected with Schistosoma
mansoni or the filarial worm Dipetalonema viteae (Capron et a1,

1972). Early stage Trichinella spiralis infections in mice produced
immunosuppression that has been characterized by Faubert and Tanner
(1974) and Cypess et a1 (1973), and which is passively transferable
with infected mouse serum. Depression of primary and particularly
secondary antibody responses to sheep red blood cells occurs both
acutely and chronically in infection with the larval stage of the

tapeworm, Taenia crassiceps (Good and Miller, 1976).

II. ROLE OF COMPLEMENT IN SPECIFIC AND NON-SPECIFIC IMMUNITY

The mechanisms reviewed thus far for immune avoidance by
helminths or protozoa attempt to account for evasion of specific
effector mechanisms mediated via antibody production or cell-mediated
immunity. Little attention has been given to non-specific resistance
mechanisms although it is clear that these are an important component
of the host-parasite relationship. Non-specific 1ytic effects
initiated by the alternative pathway of the complement cascade
have been proposed as a key factor in host-resistance to certain
infectious agents (Pillemer et a1, 1956).

Research on this type of immunity has not kept pace with
advances in specific reactivity and the phenomena have often been
classified as comprising "innate" resistance. However, it has
become clear that in some cases the success or failure of an infectious
agent may reflect a balance between parasite resistance and host
rejection mechanisms which revolve around complement activation
at the host-parasite interface. This activation of complement
can occur by either antigen specific or non-specific initiators.

For most parasites the limiting surface which contacts the host is
a membrane which can be shown to be susceptible to 1ytic effects,
and therefore interactions between infectious organisms and this

host amplification system will be reviewed in some detail.

a. Complement fixation

 

In recent reviews by Fearon and Austen (1976) and Fearon et
a1 (1974), a detailed description of the alternate pathway for
complement activation and the enzymes involved is provided. These

authors also reiterate the evidence for the existence of the

9

alternate pathway as distinct from the classical pathway. Two

of the most critical developments were: a) the purification of

a protein, termed properdin, from normal serum by Pensky et al

(1968) which was not immunoglobulin, yet was capable, when reacted
with certain polysaccharides or particular bacterial, viral, protozoal,
or fungal organisms, of activating complement and neutralizing or
lysing the organism (Pillemer et a1, 1954); b) the discovery of the
ability of cobra venom factor to react with non-classical complement
proteins to generate complement-consuming activity (Gotze and
Muller-Eberhard, 1971).

Although the activation of properdin by a number of complex
polysaccharides (e.g. lipopolysaccharide from Gram-negative bacteria,
zymosan, dextrans) initiates a pathway for C3 convertase formation
and a C3b-dependent amplification loop for more C3 cleavage, this
pathway is relatively inefficient in comparison with the classical
pathway in C3b generation (Brade et a1, 1973). This is true only
for the initial C3b generation since activated properdin seems to
greatly facilitate cleavage of C3 by the amplification loop once
it is initiated (Fearon and Austen, 1977). The zymosan—induced
properdin-dependent cleavage of C3 is impaired by diluting the
serum (Fearon and Austen, 1976).

The biologically important effects on foreign organisms
which are properdin-dependent probably require the generation of
a 1ytic complex composed of the terminal complement sequence CS
through C9 at a surface membrane. This sequence is started by
cleavage of C5 to form C5a and C5b. C5b is labile and must rapidly
join with C6 either on a membrane or in solution in order to form

a C567 complex which is cytolytic. Enhanced cytolytic activity

10

of membrance bound C567 is acquired with binding of C8 and C9 to

 

form C56789. The alternative pathway enzyme required for cleavage
of C5 is formed by C3b binding factor B in the presence of factor
‘5 to form C3B which then cleaves C3 into C3a and binds C3b. This
complex, GEES} is able to cleave C5 to CSa and C5b. The limiting
factor in alternative pathway generation of C3 convertase activity,
CSBB, and the subsequent formation of 1ytic complexes is the initial,
properdin-dependent formation of C3b from C3 (Fearon and Austen, 1976).
However, after C3b is generated by the inducing action of properdin
on factors D and B, or after it is generated by classical pathway
activation, then properdin markedly enhances the C3b-dependent
amplification loop which produces C353 (Fearon and Austen, 1977).
Thus serum which is diluted is impaired in cytolytic activity when
C3b generation is restricted to properdin induction; however,
cytolytic activity is unimpaired if C3b is provided. Indeed,
active properdin (generated by incubation with zymosan) greatly
enhances cleavage of C3 when C3b is provided.

Lipopolysaccharide (LPS) and zymosan, which are typically
associated with alternative pathway activation, have been shown
to utilize Ca++-dependent mechanisms (possibly the classical pathway)
which enhance C3 conversion. C3 conversion independent of Ca++
was approximately 60-70% of C3 conversion in the presence of both
Mg++ and Ca++ (Snyderman and Pike, 1975). If the generation of
C35; activity is critical to the cytolytic effects on bacteria,
viruses, and protozoa which are attributed to the alternative
pathway by Pillemer et a1 (1954), then the limited activation to
the classical pathway by materials which may be present on these

infectious agents, would greatly enhance CBBb formation by supplying

ll
limited amounts of C3b in the presence of active properdin for a
fully active amplification loop.

The non-specific generation of cytolytic activity is typically
attributed to the alternative pathway when this activity can be demon-
strated without antibody-antigen interaction or in the absence of
Ca++, or when early classical complement components are not required
or consumed. The specific generation of cytolytic activity as
reviewed by Fearon and Austen (1976) requires initiation of the
classical pathway by complement—fixing antibody-antigen complexes.
The first complement component, C1, binds these aggregates of
certain classes of antibody (only IgGl, IgG2, IgG3 and IgM in
humans). This binding alters the Cl component so that one of its
subcomponents, C13, is then able to cleave complement proteins
C4 and C2 with the subsequent formation of CIZEthich is a C3
convertase analogous to C3BbI The 1ytic complex formation mechanism
which follows C3 conversion is identical for both the classical and
alternative pathways, i.e., C5 cleavage and C56789 formation.

Though the classical pathway for complement-dependent lysis
is usually considered only as a specifically directed 1ytic activity,
this pathway can be activated non-specifically (without antibody-
antigen complexes) by other molecular interactions such as binding
in the presence or absence of C-reactive protein. Rent et a1
(1975) demonstrated that when protamine (polycation) and heparin
(polysulfated polyanion) in the proper proportion were incubated
together in the presence of serum, complement components C1, C4,

C2, and C3-9 were consumed. This consumption of complement components
was also shown to occur when interactions between polyanions, such

as DNA, chondroitin sulfate, and dextran sulfate, and either protamine

12
sulfate or poly-L-lysine where carried out in the presence of
C-reactive protein (CRP) (Claus et a1, 1977).

CRP is a serum protein found in increased levels during the
acute phase of inflammatory conditions. It was originally detected
by its ability to precipitate pneumonococcal C-polysaccharide
(Abernethy and Avery, 1941); this activity is now known to be due
to a binding capacity for polycations (Siegel et a1, 1975). CRP
binding with pneumococcal C-polysaccharide or with the choline
phosphatides, lecithin and sphingomyelin, initiates the classical
pathway (Kaplan and Volanakis, 1974) and CRP in small amounts greatly
enhances classical complement consumption by interactions between
polyelectrolytes. These reactions are all physiologically feasible
in that the amounts of CRP required are similar to those detectable
during inflammatory conditions and polyelectrolytes such as heparin,
DNA, chrondroitin sulfate and lysozyme commonly occur where cells
are being destroyed (Claus et a1, 1977).

When polycations are not present during incubation of certain
polyanions (heparin, DNA, dextran sulfate, and cellulose sulfate)
with serum, the effect is C1 depletion and consumption of C3-9
while C4 and C2 are not consumed (Rent et a1, 1975). The consumption
of C3-9 by certain polysaccharides and certain polyanionic, heparin-
1ike mucopolysaccharides, including heparin, is believed to be the
result of induction of the alternative pathway (Loos et a1, 1974).

In summary then, some of the non-specific activators of
complement are: 1) many polysaccharides, both neutral and negatively
charged, which are able to activate the alternative pathway, 2)
certain polyanions, including some polysulfated polysaccharides

which are able to suppress Cl activity by enhancing a naturally

13
occurring inhibitor of C1 (Rent et al, 1976), 3) interacting poly-
electrolytes which in certain proportions can activate the entire

classical pathway. This activity is greatly enhanced by CRP.

b. The involvement of complement in resistance to parasites

 

There is ample evidence that 1ytic effects on protozoan and
helminthic parasites do occur but until recently they have not
been examined critically in the light of experiences with bacterial
organisms. Both non-specific lysis and specific lysis have been
described and persistence of organisms in the host probably depends
on avoidance of surface complement fixation in some manner” Clearly
it is not possible to review this aspect of the whole spectrum
of host-parasite relationships, but several important diseases
have been investigated in sufficient detail to provide some basis
for discussion. The trypanosomes, schistosomes and taeniid cestodes
have been selected for this purpose.

In the genus Trypanosoma, circulating parasites have been
shown to interact with complement in several ways. These hemo-
flagellates are vector borne and are responsible for several important
disease syndromes in man and animals. In South American trypanosomes
there is both an intracellular phase in macrophages and a circulating
blood plasma phase. The role of humoral immunity in T. cruzi
infection (Chagas' disease) has only recently begun to be investigated
(Gable, 1970 and Budzko et a1, 1975).

Early evidence from the clinical observation that blood stream
forms of T. cruzi could be isolated from humans and experimental
animals with circulating antibodies to the parasite (Romana, 1963;

Cerisola et al, 1969; Pizzi, 1957) suggested that it was resistant

14

to immune lysis. Later in in vitro experiments parasites were
observed directly and there was no evidence of immune lysis of blood
stream forms (Teixeira, 1974). However, other studies on experimental
infections in mice showed that if complement was depleted in infected
mice parasitemia levels rose more rapidly, survival times were
shortened and greater numbers of parasites developed. This was
particularly true if complement depletion was performed at the

stage of infection when blood stream forms were emerging from
macrophages (Budzko et a1, 1975). These workers also established
that blood stream forms could be lysed in vitro in hyperimmune

mouse sera as long as complement was present (Budzko et a1, 1975).
Whether or not the parasite is susceptible to lysis in vivo is

still debatable. It may be that this parasite in one or all of

its forms is able to co-exist with complement and antibody in
certain mammalian hosts. This may be supported by the observation
of Anziano et a1 (1972) that culture forms of T. cruzi (mainly
epimastigotes) are lysed by antibody and complement. The fact that
T. cruzi is a more acute disease in children than adults and that
certain species of animals may be naturally resistant due to non—
specific complement-dependent lysis mediated by the alternative
pathway (Kierzenbaum et a1, 1976) suggests that the complement

level is involved in avoidance of immune lysis by the parasite.

In African trypanosomiasis parasite numbers in the blood
characteristically fluctuate and this pattern has been correlated
with the appearance of new surface antigenic determinants and the
development one week later of specific (IgM) antibodies (Seed et
al, 1969). Unfortunately there are no reports in which the role

of complement has been studied in vivo in the humoral immunity

15

demonstrated in African trypanosomiasis. Immunoglobulins IgM and
IgG have been shown to be responsible for passive immunity (Seed
and Gam, 1966; Seed, 1972, 1977) and the appearance of new antigenic
variants in relapse parasitemias (Luckings, 1974; Takayanagi and
Enviguez, 1973). The African trypanosomes are entirely extracellular
and regularly occur in the blood stream in high numbers causing a
long term chronic disease, and it seems likely that they have an
ability to evade immune lysis even in the presence of antibodies to
specific or non-variant antigens (Losos and Ikede, 1972).

There is evidence for a 1ytic effect of serum in schistosomiasis.
Schistosoma mansoni in the adult form is a small trematode which
has been found to exist for more than twenty years in the lumen of
the hepatic portal veins of man. It is estimated that more than
200 million people are hosts of this chronic infection. A variety
of other Schistosoma species infect the blood vessels of man and
other mammals. Cercaria released from the snail, the intermediate
host, enter the primary host by skin penetration. Cercaria, immed-
iately upon penetration of the primary host, undergo drastic morph-
ological changes, including shedding and replacement of the surface
(tegument). The early stage migrating form, prior to the adult
sessile stage, is called a schistosomule and it is during this
stage within 96 hours of host penetration that the parasite is
most likely to be destroyed by the immune response, particularly
if it has entered a previously sensitized host (Clegg, 1974).
Adult schistosomes of a primary infection or even adult schistosomes
transplanted into a sensitized host (Hockley and Smithers, 1970)
are resistant to immune rejection though they effectively immunize

the host against the invading schistosomule.

16

Many investigators have postulated that the appearance of
host antigens on schistosomules during the early invasive stage
is the critical immune evasive mechanism (Clegg, 1974; Smithers,
1972). However, Dean (1977) has shown that this may not be the case
and that schistosomules which develop for 96 hours in vitro in the
absence of host antigens are still capable of immune avoidance.

Indirect evidence from in vitro studies indicates that complement
is required for the parasiticidal effects of normal immune sera
on cercariae and schistosomules of Schistosoma mansoni. Standen
(1952) and Kagan and Levine (1955) demonstrated that normal sera
from a variety of mammals had cercaricidal activity in vitro which
was heat labile (56°C for 30'). When sera from these animals after
hyperimmunization were reacted with cercariae in vitro the parasites
were immobilized by precipitin formation, but they were not killed.
The interaction of cercariae with normal serum with the resulting
cercaricidal effects has been shown to be complement-dependent
and generated mainly through the alternative pathway (Machado et
a1, 1975). This interaction of cercariae with normal serum resulted
in the generation of anaphylatoxin. Schistosomula which lack the
external membranes of cercariae are not killed by normal serum
(Capron et a1, 1974). However, early stage (24 hour) schistosomula
incubated with serum from a naturally or experimentally infected
host are killed in the presence of heat labile serum factors (Capron
et al, 1974). Later stage (4 days) schistosomula are much more
resistant to complement-mediated antibody-dependent lysis (Clegg
and Smithers, 1972). Von Lichtenberg et a1 (1977) suggested that
these schistosomula non-specifically activate complement. Adult

schistosomes incubated in fresh serum from an infected homologous

17
host do not suffer damage; however, adult schistosomes incubated
in fresh serum containing antibodies for host antigens, which are on
the parasite surface, suffer tegumental damage but are not killed
unless activated peritoneal macrophages are present (Perez and
Terry, 1973).

Clearly complement plays a critical role in immune damage
or lysis at each stage of development of Schistosoma mansoni in
mammalian hosts. Each stage avoids immune damage with varying
success depending on whether or not the host has been sensitized
to the parasite and on the ability of later stages to counter
complement-dependent immunity. Recent reports indicate that adult
schistosomes are capable of avoiding complement-dependent immunity
even without the presence of host antigens on their surfaces (Dean,
l977),though the mechanism of this is unknown.

There is a similarity between early infectious stages of
Schistosoma sp and the metacestodes of Taenia taeniaeformis with
respect to complement-mediated immune rejection. T. taeniaefbrmis
exists as an adult in the lumen of the small intestine of cats
and inhabits the liver tissue of rodents in its larval form.

Gravid segments from adult tapeworms, when passed in the feces

of cats, contain eggs which upon ingestion by rats become infective
embryos in the rat small intestine. These embryos pass through

the intestinal epithelium and are subsequently carried to the liver
by the portal circulation. Within a few hours of egg ingestion,
invasive embryos are found migrating in the liver parenchyma and
continue to do so until by 3 days they begin to form spherical
cysticerci. The fluid filled cysticercus enlarges from about 15

to 250 u in diameter at six days post-infection. During this time

18
the cellular activity around the parasite is complex, but a partic-
ularly prominent eosinophil infiltration occurs by about 10 days
which continues until about 35 days post-infection. During the
second week of infection a layer of fibroblasts begins to surround
the cysticercus which becomes a thin fibrous capsule around the late
larval stage.

Campbell (1932) demonstrated a passively transferable humoral
immunity for T. taeniaefbrmis in rats where serum from an infected
rat was effective in killing invasive or developing larvae to a
gradually decreasing extent during the first 10 days post-infection.
Leid and Williams (1974) found that the passively transferable
humoral immunity which developed in rats during the first 4 weeks
of infection was due to antibodies of the IgG2a immunoglobulin
class. The Inga class of immunoglobulins of the rat fixes complement
and the identification of complement as the required mediator for
the protective effect of immune rat serum was determined by Musoke
and Williams (1975) both in vitro and in vivo. Developing larvae
up to four days post-infection were completely susceptible to
complement-dependent immunity, but by 10 days parasites were no
longer affected. Between 4 and 8 days post-infection the larvae
showed intermediate degrees of resistance. This acquisition of
resistance to complement-mediated immune destruction during the
initial days post-infection closely parallels the chronology of
resistance development by schistosomules.

In addition to the similarities of immuneaavoidance in early
invasive stages between S. mansoni and T. taeniaefbrmis further
parallels can be drawn between adult S. mansoni and fully developed

larval T. taeniaefbrmis by examining the interaction of these

19

stages with immune serum and complement. Adult schistosomes experience
tegumental coat perturbation but not death when exposed to hyperimmune
serum. Similarly Hustead and Williams (l977a,b) have shown that
resistant late stage cysticerci incubated in immune rat serum
transiently lost membrane permeability control for small proteins

such as ribonuclease but remain able to exclude bovine serum albumin.
This disruption in functional membrane integrity is rapidly corrected
by the parasite within an hour while still in the presence of immune
rat serum, during which complement levels in the serum were greatly
depleted. Thus both parasites have some ability to compensate for

the destructive action of antibody and complement to which they

were subjected in in vitro experiments, although the mechanisms

by which they do so are not clear.

c. Resistance to lysis in living cells

 

The above discussion of lysis in relation to parasites provides
some grounds for believing that organisms surviving in tissues can
develop to the point where they may no longer be susceptible to
complement attack, and that surface membrane factors may be involved
in the avoidance mechanism. In fact,the standard assay for activation
of the complement cascade to completion is based on the lysis of
aged erythrocytes which are dead cells bounded by a bilaminate
membrane. Extrapolation of the mechanism of cytolysis to cells
actively metabolizing and capable of membrane repair must be done
with caution. There is evidence that in various cell types surface
complement-fixation does not necessarily lead to cell lysis or cell
death, and the mechanisms whereby this comes about may be important

in evasion of immune attack by parasites.

20

Certain strains of Gram-negative bacteria differ only in
their resistance to complement-mediated lysis. Two Gram-negative
bacteria, Salmonella typhimurium and Escherichia coli have been
extensively studied for resistance to complement-mediated lysis
and for virulence as disease organisms. The non-virulent, serum-
sensitive M206 strain of Salmonella typhimurium is a rough strain
which is identical to the virulent, serum-resistant, C5 strain of
S. typhimurium except that C5 is smooth. Similarly S. typhimurium
LT2, S. minnesota 218s and S. enteritidis 795 are all smooth strains
with complete resistance to the bactericidal action of complement,
while the parallel rough strains are lysed in serum (Reynolds and
Pruul, l97la,b). E. coli which differ only in the amount of
lipopolysaccharide (LPS) in their surface coat are either resistant
or sensitive to complement lysis (Glynn, 1972). Smooth strains of
Gram— negative bacteria carry greater amounts of LPS on their
surfaces compared to rough strains. The amount of LPS on the bac-
terial surface is directly proportional to complement resistance
(Glynn, 1972). This LPS is highly active in depleting hemolytic
complement levels by initiating the complement cascade (Gewurz
et a1, 1968).

The deposition of C56789 and the formation of complement
"lesions" on the LPS material extracted from both smooth and rough
Salmonella minnesota as well as on the surface of both strains of
the whole bacterium (Mergenhagen et a1, 1968) indicates that the
90A doughnut-shaped electron-dense rings seen on SRBC membranes
after complement-mediated lysis (Borsos et al, 1964) do not nec-
essarily induce lysis of bacteria when formed on the LPS surface

coat .

21

Experiments with mammalian eukaryotic cells have provided
the most direct evidence to date that the formation of antibody-
induced complement "lesions" at or near the cell membrane does not
necessarily lead to cell lysis or death. Cikes (1970a,b; 1971)
reported that virus-infected cells were susceptible to complement-
mediated, anti-viral antibody lysis only during the stationary
phase of growth in culture, but were resistant to lysis during the
logarithmic growth phase. These workers suggested that antigenic
sites were not exposed during logarithmic growth, but this was
shown to be incorrect when Lerner et a1 (1971) demonstrated that
viral antigen was present on the cell surface, accessible to antibody,
and able to activate complement in the presence of antibody throughout
all cellular growth phases. They confirmed that cytotoxicity was
limited to the G1 phase of cell growth, but all nine complement
components were consumed and C3 and C4 were bound to lymphoma cells
in the presence of antibody during all phases of cell growth.
Further quantitative studies by Cooper et a1 (1974) demonstrated
that equal amounts of C5 and C8 bound to antibody-treated cells
in all stages of growth, and that electron dense circular areas,
typical of terminal complement "lesions", appeared on the surfaces
of cells at all stages of growth when exposed to complement and
antiviral antibody.

The ability of cultured cells of mammalian origin to resist
lysis in the presence of specific antibody and complement during
S phase of growth has been demonstrated for murine tumor lines
YCAB (Lerner, 1971), YAC (Cikes, 1972), L1210 (Gotze, 1972) and
chinese hamster lung cells (Shipley, 1971). The human lymphoid

cell line RPMI8866 was shown by Pellegrino et a1 (1974) to be

22
similarly variable in its susceptibility to complement and antisera
directed against histocompatibility antigens, even though the
quantitation of antigens, bound antibody, and bound C3, C4, and
C8 per cell surface area showed no variability during the cell
cycle. Pellegrino et a1 (1974) concluded that differential sus-
ceptibility to lysis was due to changes in cell surface properties
during the cell cycle, other than those associated with antigenic

expression.

23

III. MECHANISMS OF RESISTANCE TO LYSIS

a. Tissue location

 

Whether or not growth-related surface properties are involved
in resistance to complement by replicating or developing successful
tissue-dwelling parasites remains to be seen. There is presently
insufficient information on the differential susceptibility to
lysis in parasites, comparable to that which has been derived for
bacterial and mammalian cell systems. In order to relate these
observations to evasion of immunity by organisms in tissues it is
relevant that hemolytic complement titres are not identical throughout
all host tissues.

Tissue location is certainly an important determinant of
survival in bacteria. Complement-sensitive strains of E. coli
may inhabit the gut lumen, but complement-resistant strains differing
only in the amount of K antigen on their surface are able to survive
in the circulatory system (Roantree and Rantz, 1960; Roantree and
Pappas, 1960). Evidently the effector mechanism is not able to
affect lumen-dwelling organisms. Host encapsulating reactions
may serve to limit the access of large molecular weight humoral
components to tissue-dwelling sessile parasites (Campbell, 1938),
and recent work on measles supports this notion. Tissue sites
distant from the plasma may be particularly deficient for immune
responses requiring the functional alternative pathway, as postulated
by Joseph and Oldstone (1975) for virus-infected cells in the central
nervous system. Joseph, et al (1975) have demonstrated that lysis
of measles virus-infected cells requires specific human IgG antibody

and the components of the alternative pathway. Thus dilution of

24
serum, perhaps in conjunction with other mechanisms which selectively
block the alternative pathway, may obviate IgG-antibody lysis of
measles virus-infected cells. Trypanosoma Vivax and brucei multiply
in the connective tissue and may thereby avoid high blood levels
of complement during cell cycle stages which may be more susceptible

to lysis.

b. Surface coat characteristics in bacteria

 

In many cases, however, accessibility of plasma factors is
clearly not an issue and parasites flourish even though they confront
complement in the blood at many stages in their life cycle. It
seems more likely that some surface component or components are
crucial in effecting evasion. The background of information on
bacterial surface characteristics provides support for this proposal.
In the case of the pneumococcus the thick layer of polysaccharide
molecules which is outside the limiting cell membrane is thought to
restrict the penetration of large host molecules by the highly
hydrated state of the saccharide matrices (Glynn, 1972; Apffel and
Peters, 1970). Common examples of hydrated saccharide matrices
or gelification excluding proteins are Sephadex gel filtration
beads and hyaluronic acid exclusion of protein (Ogston and Preston,
1966; Laurent, 1963). In a similar manner, the cell wall of
Mycobacteria sp. has been proposed to function as a protective
inert barrier (Glynn, 1972).

More definitive experiments with serum-resistant Gram-negative
bacteria have demonstrated that the lipopolysaccharide surface
coat of smooth strains is required for complement resistance (Dlabac,

1968; Sterzl, 1964). Reynolds and Pruul (l97la,b) have shown that

25

smooth strain Salmonella are insensitive to the action of specific
antibody and complement in the presence of Mg++ or saline but are
lysed if monovalent cations or low concentrations of EDTA are
substituted or added. The data were interpreted as indicating that
divalent cations such as Mg++ or Ca++ are required for the structural
integrity and bridging of the polyanionic molecules composing LPS.
Endotoxic LPS of Salmonella activates complement and binds terminal
complement components (Mergenhagen et a1, 1968), yet antibody has
been shown to penetrate to the plasma membrane level without lysis

in the presence of complement, unless monovalent cations or chelators
are present, in which case bacterial lysis occurs (Reynolds et a1,
1975). Two mechanisms have been proposed to account for the results
of these experiments; activation and decay of complement components
at loci distant from critical membrane sites, and inhibition of

activated C5 binding by LPS (Reynolds et a1, 1975).

c. Surface polyanions and their potential role in resistance in lysis
Unfortunately no direct evidence exists to explain the phenomenon

of cell cycle variation in susceptibility to complement-mediated

lysis for tumor and transformed cells of mammalian origin. However,

a correlation does exist between diminished susceptibility to

lysis and the presence of heparan sulfate (sulfated polyanion)

on the surface of cultured cells (Kraemer and Tobey, 1972). Heparan

sulfate and other polysulfated molecules are known to interact with

the complement proteins. Heparan sulfate is synthesized intra-

cellularly and appears in the surface coat of many different mammalian

cell types, and premitotic loss of heparan sulfate occurs during

the cell cycle of cultured cells (Kraemer and Tobey, 1972; Chiarugi,

26

1974) and in the regenerating rat liver (Kojima and Koizumi, 1974).
The highest levels of heparan sulfate occur on the cell surface

during the S phase of the cell cycle which corresponds to the stage

at which virus-transformed cells and leukemia cells are most resistant
to complement lysis (see section II, c).

More direct evidence that sulfated polyanions or acid mucopoly-
saccharides play a role in resistance to lysis was obtained when Lippman
(1968) treated Moloney-induced tumor cells with various acid mucopoly-
saccharides prior to injection into allogeneic hosts or before incubation
with antisera to H-2 antigens or viral antigens plus complement. In her
experiments, enhanced tumor growth and resistance of tumor cells to com-
plement-mediated immune lysis was observed in those cells pretreated
with acid mucopolysaccharides (heparin, keratin sulfate and, to a
lesser degree, hyaluronic acid).

Chiarugi et a1 (1974) have speculated that surface heparan sul-
fate is a control element in regulating cell mitosis and differentiation.
Such a role for cell surface heparan sulfate would have important impli-
cations in cell-mediated immunity as well as humoral immunity. A
limited number of reports on the effects of heparan sulfate upon cell-
mediated immunity have been published, and even though this review has
been mainly concerned with humoral immunity, these results emphasize
the potential consequences of polyanionic polysaccharides on immune
reactions. Viklicky et a1 (1974) showed that long term treatment of
recipients of skin allografts (with weak non-H-Z histocompatibility
barrier) with small doses of chondroitin sulfate results in 19%
successful grafts compared to no successful grafts in the controls.
Further studies by Viklicky et a1 (1975) demonstrated that if chondroitin

sulfate or heparin was present in the reaction medium for a complement-

27

independent cytotoxic reaction the percentage of cytotoxicity was re-
duced to control levels (non-sensitized cells and killer cells. In this
same paper antibody-induced redistribution or capping of H-2 antigens on
lymph node cells was found to be greatly accelerated when these cells
were pretreated with chondroitin sulfate or heparin. Pokorna (1976) ex-
tended this experimental design to show that chondroitin sulfate inhib-
ited FHA-induced stimulation of lymphocytes: 67% of control cells were
stimulated compared to 24% of chondroitin sulfate-treated cells.

There is evidence that some other immunologically important
events in cells are relatable to surface polyanions. Membrane
antigenic modulation by capping, shedding and pinocytosis are
thought to be influenced by surface acid mucopolysaccharides through
their role in control of Ca++ flux (Viklicky et a1, 1975; Pokorna,
1976; and Vlodavsky and Sachs, 1977). There is recent evidence
for this role in pinocytotic processes (Josefsson, 1975). These
observations and other known properties of polyanions provide
some grounds for proposing that they are important in determining
susceptibility of cells to host attack mechanisms.

The potential for polyanion interaction with complement has
been referred to above (see section II, a). The ability of an
outer coat material to trigger complement non-specifically at sites
distal from the labile membrane could serve to consume complement
and maintain local microenvironment titers at low levels. Ramif—
ications of the enhancement of Cl-INH by polyanions in terms of
their effects on a variety of components of inflammation have not
been investigated (e.g. CleINH has effects on plasmin, kallikrein,
Hageman factor, Hageman factor fragments and coagulation factor XI

(Austen, 1971)). Surface polyanions could contribute to steric

28
exclusion of macromolecular proteins by matrices of cross-linked
polysaccharides (for review see Laurent, 1977). The presence of
such a surface molecular seive could permit exclusion of large
proteins, such as Clq.

Electrostatic interactions between negatively charged outer
coat polysaccharides and host proteins could result in repulsion
or binding. Repulsion of other negative charges has been proposed
as a means of preventing interaction between maternal cells and
the glycosaminoglycan coated surfaces of mammalian trophoblast
cells of the embryo and fetus (Calatroni and Ferrante, 1969; Bill-
ington, 1975). Polyanion binding of inflammatory mediators such
as histamine could neutralize its local effects. Heparin is the
natural "counter-ion" for the histamine stored in mast cells

(Jaques, 1975).

d. Parasite surface characteristics and evidence for the presence

 

of_polyanions

 

It is clear from the literature reviewed so far that a corre-
lation has been established between resistance to complement cytolysis
in vitro in tumor and virus-transformed cells from a variety of
mammalian tissues and the levels of surface polyanions, and that
there is limited evidence that exogenously provided acid mucopoly-
saccharides confer resistance against host immune rejection.

Although comparable experimental work has not been done for parasitic
organisms there is developing evidence that many parasites have
substantial amounts of polyanionic substances in their surface coats,
and partial characterization has been achieved in several cases.

Histochemical studies have shown that both stercorarian

29

trypanosomes such as T. lewisi and T. cruzi (Dwyer, 1975 and 1976;
DeSouza and Meyer, 1975) and salivarian trypanosomes such as T.
Vivax and T. brucei (Allsopp and Njoger, 1974; Wright and Hales,
1970) contain polyanionic polysaccharides either in the surface
coat or between the surface coat and the plasma membrane. Employing
selective lectins, Dwyer (1976) has found these polysaccharides
in T. lewisi to contain N-acetyl-D-galactosamine, N—acetyl-D-glucos-
amine and possibly mannose. In his experiments culture forms of
the parasite showed less affinity for each of these lectins than
did blood stream forms. Alves and Coli (1975) identified glucosamine,
galactose, glucose and mannose in the surface glycoprotein of
T. cruzi.

In contrast to T. cruzi the lectin-binding amino sugars of
the blood stream forms of African trypanosomes are not detectable
unless a superficial glycoprotein layer is removed by protease
digestion. These glycoproteins may constitute the variant or
exoantigens which characterize the fluctuating parasitemia of
T. brucei infections (Allsopp et a1, 1971). The fact that exo-
antigens are released from the surface coat at low pH suggests
that there is non-covalent binding to the saccharide coat. This
may be similar to the electrostatic binding of glycoproteins to
heparin (Laurent, 1977). If a polyanionic polysaccharide layer
is interposed between the trypanosome limiting membrane and the
highly immunogenic variant antigens (Vickerman, 1974) then the
polysaccharide coat may be facilitating an energy conservative
flux of proteins without exposing more critical invariant antigens
of the membrane to immune attack. Indeed, T. vivax with a much

less compact surface coat has been shown to adsorb host serum

30

proteins on to its surface (Desowitz, 1970).

Electron microscopic studies by Vickerman (1974) point to
a common feature for T. brucei, T. congolense, and T. vivax involving
appearance of a surface coat on infective forms borne by arthropod
vectors. In the last stage of development in the insect vector,
just prior to infecting a mammalian host, African trypanosomes
acquire a surface coat external to the plasma membrane which they
maintain as blood stream forms in the mammalian host. This surface
coat is known to be lost when blood stream forms enter the insect
vector, though no information is available concerning its presence
on extravascular phases of T. brucei, and vivax during replication.
Allsopp and Njoger (1974) analyzed surface glycoproteins from blood
stream T. brucei and detected D-glucosamine, D—galactose and D-mannose.
Unfortunately none of the chemical analyses of Trypanosoma (of
either type) surface carbohydrates included assays for sulfate
groups. Thus the electron microscopic visualization of acidic
polysaccharides on both types of trypanosomes by ruthenium red
(Luft, 1971) and colloidal iron can only be circumstantially confirmed
by the chemical evidence for hexosamines and galactose, which are
the monosaccharides (along with uronic and iduronic acids) making
up acid mucopolysaccharides or glycosaminoglycans (Lindahl et a1,
1977).

Another protozoan, Leishmania donovani, which encounters
two distinct environments in its mammalian host has two distinct
surface types. Intracellular forms (amastigotes) have no apparent
surface coat material whereas extracellular forms (promastigotes)
have densely staining coats of negatively charged polysaccharides.

Cultured forms of amastigotes, developing into promastigotes,

31
acquired this same surface coat within 7 hours (Dwyer et a1, 1974).

In reviewing the literature on parasitic helminth surfaces,
it is apparent that there has recently developed an awareness of
the dynamic state of tegumental structures. In an extensive review
of surface ultrastructure and cytochemistry by Lumsden (1975) the
concept of an inert surface "cuticle" is dismissed and evidence
is presented on the transport of nutrients across the tegument and
on the mechanisms of avoidance of host proteolytic enzymes.

The immune susceptible early stage schistosomule and the
immune-resistant adult schistosome of S. mansoni offer possible
insights into the role of surface acidic proteoglycans or muco-
polysaccharides. Upon penetration of mammalian skin, cercariae
lose a coat of neutral polysaccharides and are left with a very
thin layer of acid mucopolysaccharides on their surfaces. The
growing schistosomula rapidly increase in surface area by adding
multiple layers of plasmalemma which possess acidic staining poly-
saccharides (Stein and Lumsden, 1973). This surface structure
is continually being replaced in mature schistosomes (Clegg, 1972)
and there is recent data to suggest that some surface component
enables the adult fluke to inhibit coagulation (Tsang and Damian,
1977). The surface material shed into the host circulation may
be the high molecular weight polysaccharide schistosome antigen
found in the blood of patients with schistosomiasis (Buck et a1,
1975). In a recent paper it has been suggested that non-specific
initiation of complement-fixation occurs around schistosomules
(Von Lichtenberg et a1, 1977).

Larval and adult stages of most cestodes have been shown

to be invested with a surface glycocalyx which is distal to the

32

plasma membrane of the tegumental cells themselves (Lumsden, 1975).
There is cytochemical evidence for the elaboration of carbohydrate
containing surface material (Oaks and Lumsden, 1971) and histo—
chemical studies have shown that tegument is the site of synthesis
.of non-glycogen polysaccharides (Lumsden, 1975), some of which have
been ultrastructurally located at the tegument surface.
Histochemically the cestode glycocalyx reacts positively
with stains for carbohydrate vicinal hydroxyl groups (Lumsden et
a1, 1970), and there is a high density of anions which Lumsden
(1972) interpreted as a reflection of the presence of acidic glycans.
Morris and Finnegan (1968) suggested that the surface of tissue-
dwelling larval cestodes of Schistocephalus solidus contained
sulfated acid mucopolysaccharides. Lumsden (1973) considered that
carboxyl groups plus inorganic acid residues also probably contributed
to the net electronegative charge on the cestode surface. While
a variety of roles have been ascribed to cestode surface acidic
polysaccharides, including participation in transport systems and
enzyme inhibition, no consideration has apparently been given to
their potential significance in the avoidance of rejection through
any of the mechanisms outlined above. The work reported in this
dissertation suggests that this possibility should not be overlooked
in the future. Recently reported observations by Kassis and Tanner
(1976) and Herd (1976) on the lytic effects of normal serum upon
larval and mature cestodes in vitro provide impetus for defining

those factors which allow parasite survival in vivo.

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ACTIVATION OF COMPLEMENT BY HYDATID CYST FLUID

OF ECHINOCOCCUS GRANULOSUS

COMPLEMENT ACTIVATION BY HYDATID FLUID

B. HAMMERBERG AND J. F. WILLIAMS

Department of Microbiology and Public Health
Michigan State University

East Lansing, Michigan 48824

This is journal article No. from the Michigan

Agricultural Experiment Station.

ABSTRACT

Factors present in hydatid cyst fluid of Echinococcus granulosus
were found to interact with complement from several species. This
non-immunologic fixation resulted in the depletion of hemolytically
active complement from fresh guinea pig serum, the conversion of
human C3 to an electrophoretically faster species and the generation
of smooth muscle contracting substances, analogous to anaphylatoxins,
in normal rat serum in vitro. Vascular permeability changes were
produced in vivo in rats and humans after intradermal inoculation
of complement interacting fractions of hydatid fluid. It is suggested
that these factors may contribute to the pathogenesis of the shock
syndrome which follows intravenous administration of hydatid fluid
in normal animals, and to the non-specificity of immunodiagnostic

skin tests for hydatid infection in man and animals.

45

INTRODUCTION

It has been known for many years that the parenteral injection
of hydatid cyst fluid in animals results in a striking sequence
of pathophysiologic changes leading to profound shock and sometimes
death (Giusti and Hug, 1923; Rocha e Silva and Grana, 1945; Tabatabai
et a1, 1973). A similar sequence of events has been described
in human patients in whom cysts of Echinococcus granulosus have
accidentally ruptured (Dew, 1928; Handjani et a1, 1969). Experimental
analysis of the shock syndrome in normal animals (Rocha e Silva and
Grana, loc. cit; Tabatabai et a1, 1973, 1974) has provided a detailed
account of the cardiovascular and respiratory responses which occur
and of their susceptibility to pharmacologic modification. Never-
theless, the underlying mechanism is not understood and although
supportive evidence is lacking, some form of immunologically mediated
anaphylaxis is generally postulated to account for the phenomenon.
Little consideration has been given to alternate explanations, but
an inherent toxicity of substances in hydatid fluid has been suggested
recently (Tabatabai et a1, 1974).

In the following study we have examined interactions between
factors present in hydatid fluid and the host complement system.
Our results indicate that these factors can initiate non-immunologic
activation of complement in normal serum and the production of
anaphylatoxins which are able to contract smooth muscle and effect
vascular permeability changes in both rats and humans. We propose
that such a mechanism may be involved in the pathogenesis of the
hydatid fluid shock syndrome and may also contribute to the development

of non-specific reactivity in immunodiagnostic skin tests.

46

47
MATERIALS AND METHODS

Experimental Animals

 

Spartan (Spb (SD) Br) rats were purchased from Spartan

Research Animals, Haslett, Michigan.

Maintenance of Parasites

 

Hydatid cysts of E. granulosus were originally derived from
secondary infections in jirds (Meriones unguiculatus) obtained
through the generosity of Dr. C. W. Schwabe, University of California,
Davis, CA. Organisms were surgically implanted into the peritoneal
cavity of rats and allowed to develop for periods of 10 to 15 months

before transfer or use (Varela-Diaz et a1, 1974).

Hydatid Fluid

 

Cysts harvested from rats were punctured and the hydatid
fluid was collected and concentrated 7X by negative pressure dialysis.
Sheep hydatid cyst fluid (SHCF) was collected by one of us (JFW)
from viscera obtained at slaughterhouses in Buenos Aires, Argentina.
Protoscolices were allowed to sediment and the clear supernatant
was removed, lyophilized and stored at -20 C for several years before

use in our laboratory.

Fractionation Procedures

 

After overnight dialysis at 4 C against 0.1 M Tris HCl buffer
pH 8.0, 5 m1 of concentrated rat hydatid cyst fluid (RHCF) was
chromatographed on a Sephadex G-200 column (90 x 2.5 cm) equilibrated
with the same buffer. The elution pattern was monitored at 280 nm.
Host serum components were removed from RHCF or chromatographic

fractions by passage over immunoadsorbent columns of glutaraldehyde

48
polymerized sheep anti-rat serum or anti-rat Fab (Avrameas and

Ternynck, 1969; Leid and Williams, 1974).

Complement Fixation

 

A slight modification of the procedure described by Kabat
and Mayer (1961) was employed for the quantitative estimation of
immune hemolysis. Sheep red blood cells (SRBC) were sensitized
with anti-serum prepared in rats by the intraperitoneal inoculation
of 108 washed cells 8 days prior to bleeding.

Quantitation of complement fixation was carried out by
measuring the number of CH/50 units remaining in 0.5 ml of normal
guinea pig serum after incubation for 1 hr at 37 C with an equal
volume of hydatid cyst fluid or chromatographic fractions. One
half ml of a suspension of sensitized SRBC (3 X 108) was added
to doubling dilutions of the incubation mixture and after an additional
1 hr at 37 C, 2 ml of ice-cold phosphate buffered saline (PBS)
pH 7.4 were added to stop the reaction. The samples were then
centrifuged at 1000 g for 10 min at 4 C and the degree of hemolysis
was determined by spectrophotometric analysis of the supernatant

at 541 nm.

C3 Conversion

 

Immunoelectrophoretic analysis of normal human serum was
conducted on 2.5 X 7.5 cm slides coated with 2% agar in 0.1 M barbital
buffer pH 8.6 containing 0.001 M EDTA. Electrophoretic separation
was continued for 2.5 hr and precipitation bands were developed using
monospecific anti-human C3 prepared in sheep. Purified C3 for the
production of this reagent was kindly provided by Dr. R. A. Patrick,

Department of Microbiology and Public Health, Michigan State University.

49
Positive control preparations exhibiting complete conversion of C3

were incubated with zymosan for 1 hr at 37 C (Muller-Eberhard and

Fjellstrom, 1971).

Anaphylatoxin assay

 

Anaphylatoxin generation for positive control reagents was
accomplished by the incubation of agar with normal rat serum (NRS)
for 1 hr at 37 C (Lepow et a1, 1969). The assay for the production
of smooth muscle contracting substances was performed on segments
of atropinized normal guinea pig ileum as described by Dias da
Silva et a1 (1967). Histamine (Sigma Chemical Co., St. Louis, MO)
was used as a standard for the detection of smooth muscle responsive-
ness. Mepyramine maleate (Merck, Sharp and Dohme, Research Labor-
atories, Rahway, NJ) (0.05 ug) was added to the tissue bath when
antihistamine treated preparations were required. Bradykinin was

also purchased from Sigma Chemical Co.

Vascular permeability changes in skin

 

Normal female rats were shaved over the dorsal region and
given intravenous (i.v.) doses of 0.5 m1 of 1% Coomassie sky blue
10 min prior to the intradermal inoculation of 0.05 ml of samples
to be tested. Vascular permeability changes were manifested by
the extravasation of dye over the test sites and the reactions were
recorded 10 min after inoculation. Histamine (10 ug) was used to
provide a positive control. Diphenhydramine HCl, mepyramine maleate
or cyproheptadine treatment of rats at 20 mg/kg i.v. 15 min prior
to the test was used to inhibit histamine mediated reactions.
Intradermal inoculations of 0.05 ml quantities of hydatid

fluid fractions were made in the skin of the forearm of 3 human

50
volunteers. Maximum diameters of edematous wheals were recorded

10 min later.

RESULTS

A profound depression of complement levels in normal guinea
pig serum (NGPS) resulted from incubation with an equal volume of
either RHCF (7X conc.) or SHCF (10X conc.). CH/50 levels in NGPS
after incubation with RHCF were consistently reduced to 80 units
or less while control preparations incubated with buffer maintained
values of 160 units or above. Reductions with SHCF were slightly
greater.

Double immunodiffusion tests showed that RHCF contained several
host serum proteins. These were removed by passage over an immuno-
adsorbent column of sheep anti whole rat serum (Fig. 1). Removal
of host components from cyst fluid did not affect its capacity to
reduce complement levels in NGPS. After gel filtration of RHCF,
the major portion of the antihemolytic activity was present in the
initial void volume peak. Rat IgM was removed from the fraction
by passage over an immunoadsorbent column of sheep anti-rat Fab.
The complement fixing activity of the fraction was retained and
this preparation, designated RHCF-1, was used in the biological
assays detailed below.

The capacity of RHCF and SHCF to deplete complement levels
was associated with their ability to cause the conversion of C3
to an electrophoretically faster migrating species. For this
test SHCF or RHCF was incubated with an equal volume of normal
human serum (NHS) for 1 hr at 37 C. Control preparations consisted

of NHS incubated with PBS or NHS incubated with an equal volume

51

Figure 1. Ouchterlony analysis of rat hydatid fluid (HF)
before and after absorption of host serum proteins (HF Abs).
Anti whole rat serum was placed in the center well.

Figure 2. Immunoelectrophoretic analysis of human serum
following incubation with either hydatid fluid (top) or zymosan
(bottom). Normal serum was used as a control. The troughs contained
sheep anti-human C3.

 

—“ —- — —.‘.—'_“.—_——— -— W

--—— —- “r“

- _—.—. __,_. ._ _

 

53
of zymosan (15 mg/ml). After electrophoresis and development with
anti-human C3 almost complete conversion was demonstrated for NHS
mixed with cyst fluids or zymosan whereas minimal degradation
occurred with NHS alone (Fig. 2).

The ability of factors in cyst fluid to generate the production
of anaphylatoxins was tested in a bio-assay for smooth muscle con-
tracting substances in vitro. Normal rat serum was incubated for
1 hr at 37 C with RHCF-l, or with agar. After centrifugation,

0.05 ml samples of supernatant sera were added to an incubation

vessel in which a strip of guinea pig ileum was suspended and

attached to a lever-arm recording device. Normal rat serum-agar
incubation mixtures are known to result in the production of
anaphylatoxins which are characterized by the following criteria:

a) they cause contraction of atropinized ileum, b) these contractions
are inhibited by pre-treatment of the muscle preparation with anti-
histamines, and c) repeated application leads to diminishing responsive-
ness and eventually desensitization (tachyphylaxis). Desensitized
preparations retain responsiveness to histamine and bradykinin.

In our experiments all these criteria were met for both NRS-agar

and NRS-RHCF-l mixtures (Fig. 3). In addition, the activity gen-
erated by cyst fluid factors was shown to be analogous to anaphyla-
toxins in that tachyphylaxis induced by NRS-agar cross-desensitized

to NRS-RHCF-l and vice versa. Mepyramine maleate pre-treatment of
smooth muscle preparations completely abolished responses to histamine,
NRS-agar or NRS-RHCF-l, but contractions were still obtained by the
addition of 0.05 ug of bradykinin to the bath.

The in vitro demonstration of C3 conversion and the production

54

Figure 3. Smooth muscle contraction in response to sequential
stimulation. Vertical bars represent height of contraction (mm)
registered by guinea pig ileum strip suspended in bath to which were
added histamine (open bar), RHCF-l plus normal rat serum (dark
stipple), agar plus normal rat serum (light stipple) or bradykinin
(diagonal hatch). The preparation was mephraminized at T .

lo.

__, CONTRACTION (an )
fl L
HISTI J

RHCF—

HISTr J

RHCF

 

 

L—

HISTF

Slel'lWLLS <—-——

 

 

 

 

 

 

 

 

 

 

 

 

 

 

56

of smooth muscle contracting substances suggested the possibility
that RHCF-l might cause changes in vascular permeability in vivo.
Rats prepared by the i.v. inoculation of Coomassie blue dye were
given intradermal doses of 0.05 ml of RHCF-1, histamine (10 ug)
or buffer diluent. Marked extravasation of dye occurred within
10 min at sites receiving RHCF-1 or histamine. Reactions reached
up to 8 mm in diameter 10 min after inoculation and tended to subside
rapidly thereafter. Control sites showed only minimal extravasation
of dye at the site of needle trauma. Administration of antihistamines
prior to the intradermal inoculations completely abolished reactivity
to histamine. However, some extravasation of dye persisted at
sites inoculated with RHCF-l although responses to this fraction
were substantially reduced.

The ability to produce vascular permeability changes in vivo
was not restricted to rats. Three normal human volunteers were
given inoculations of 0.05 ml of RHCF-l intradermally in the skin
of the forearm. Marked wheal and erythema reactions occurred in
all subjects with maximum reactivity evident by 10 min post-inoculation
(Fig. 4). In one case diffuse swelling of the site developed over
the succeeding hours and this area remained swollen and mildly painful

for 2 days.

57

Fiugre 4. Response to intradermal inoculation of 0.05 ml
RHCF-1 (rat hydatid fluid fraction) in normal human subject.
On the left (control) 0.05 ml of the buffer diluent was given.
The photograph was taken 10 min after inoculation.

 

 

59

DISCUSSION

These results indicate that factors present in hydatid cyst
fluid from rats or sheep are capable of interacting with serum
complement from a variety of species. These substances do not appear
to be host-derived since removal of rat serum proteins from cyst fluid
by immunoadsorption did not reduce the effectiveness of RHCF in
depleting complement levels in guinea pig serum. Additional indicators
of complement fixation were C3 conversion to an extent comparable
to that achieved with zymosan, and the generation of smooth muscle
contracting activity similar in its characteristics to the anaphylatoxin
produced in rat serum by incubation with agar. It did not appear that
the vascular permeability changes which were observed in vivo were
entirely attributable to anaphylatoxin production since some alterations
of permeability were effected in antihistamine-treated rats, and the
protracted course of the reaction in one of the human subjects was
not typical of histamine-mediated responses. Possibly other pathways
leading to the release of inflammatory mediators are activated
in vivo.

The mechanisms whereby complement activation is initiated
by E. granulosus cyst fluid have yet to be determined, although
recent work with bladder fluid from Taenia taeniaefbrmis, in which
comparable factors occur, suggests that the alternate or properdin
pathway is predominantly involved (Hammerberg et a1, 1976). Sufficient
evidence exists to propose that the production of factors capable of
interacting with complement in this way is a characteristic of a
broad spectrum of taeniid metacestodes, and we have speculated

that they play a crucial role in the host-parasite relationship

6O

(Hammerberg et al, unpublished; Hustead and Williams, 1976).

The manifestations of complement activation which we have
demonstrated may be of importance in the shock syndrome produced
by hydatid fluid injections in normal animals. The respiratory
and cardiovascular responses which characterize this syndrome have
been investigated by Tabatabai et a1 (1973, 1974). They showed
that pre-treatment with antihistamines abolished responses to intra-
venously administered cyst fluid in dogs, but was less effective
in sheep. Atropinization was without effect in either species and
clear evidence of tachyphylaxis appeared when dogs were given repeated
sub-lethal doses of SHCF.

Tabatabai et a1 considered that their findings were probably
the result of immunologically mediated histamine release, and that
prior sensitization to cross reacting antigens was involved in the
induction of the response in normal animals. Reaginic antibodies
are produced in both definitive and intermediate hosts which become
infected with E. granulosus (Williams and Perez-Esandi, 1971; Schantz,
1973; Dessaint et a1, 1975) and cross reacting antigens are known
to occur in closely related helminths which may parasitize animals
and man. While an allergic mechanism may indeed account for the
shock phenomenon in suitably sensitized hosts, our observations
on the interaction between parasite factors and complement suggest
that this also should be considered as a contributing mechanism
in the pathogenesis of the syndrome. Likewise, the widespread
occurrence of non-specificity in immediate hypersensitivity skin
tests for hydatid cyst infection (Varela-Diaz and Coltorti, 1974;
Cherubin, 1969) can be explained on the basis of allergic

sensitization to cross reacting antigens. However the generation

61
of anaphylatoxins in vivo by cyst fluid components cannot be ignored
as a possible element in the production of rapid wheal and flare
responses in the skin. Further studies on the pathophysiological
effects of these soluble complement-activating factors are presently
under way in our laboratory. They do not appear to be related to
the complement-fixing activity associated with insoluble calcareous
granules described by Kassis and Tanner (1976) although they may

prove to have similar roles in the host-parasite relationship.

ACKNOWLEDGEMENTS
This work was supported by U.S.P.H.S. Grant AI-10842. This
is journal article no. 7682 from the Michigan Agricultural Experiment
Station. We thank Drs. R. A. Patrick and David Liu for helpful

discussions and advice during the course of this work.

62

LITERATURE CITED

AVRAMEAS, S. and TERNYNCK, T. (1969). 'The cross-linking of
proteins with glutaraldehyde and its use for the preparation
of immunoadsorbents.‘ Immunochem. 6:53-66.

CHERUBIN, C. E. (1969). 'Nonspecific reactions to Casoni antigen.‘
Am. J. Trop. Med. Hyg. 18:387-390.

DESSAINT, J. P., BOUT, D., WATTRE, P. and CAPRON, A. (1975).
'Quantitative determination of specific IgE antibodies to
Echinococcus granulosus and IgE levels in sera from patients
with hydatid disease.‘ Immunol. 29:813-823.

DEW, H. R. (1928). 'Hydatid Disease: Its Pathology, Diagnosis
and Treatment.‘ pp. 332-330. Sydney Australasian Medical
Publishing Co.

DIAS DA SILVA, W., EISELE, J. W. and LEPOW, I. H. (1967). 'Complement
as a mediator of inflammation. III. Purification of the activity
with anaphylatoxin properties generated by interaction of the first
four components of complement and its identification as a cleavage
product of CB.‘ J. Exp. Med. 126:1027-1048.

GIUSTI, L. and HUG, E. (1923). 'Proprietes pharmacodynamiques
du liquide hydatique.’ Comptes Rendus Seanc Soc. Biol. 88:344-346.

HAMMERBERG, B., MUSOKE, A. J., HUSTEAD, S. T. and WILLIAMS, J. F.
(1976). 'Anticomplementary substances associated with Taenia
taeniaefbrmis larvae.‘ In: E. J. Soulsby, ed. Pathophysiology
of Helminth Infections. London and New York Academic Press.

(In press).

HANDJANI, A. M., FARPOUR, A., MECHANIC, K., HAGHIGHAT, A. and
DUTZ, W. (1969). 'Cardiovascular echinococcosis.‘ Am. J.
Surgery 117:666-670.

HUSTEAD, s. T. and WILLIAMS, J. F. 'Permeability studies on Taenid
metacestodes: Antibody mediated effects on membrane permeability
in larvae of Taenia taeniaeformis and Taenia crassiceps.‘ J.
Parasitol. (In press).

KASSIS, A. I. and TANNER, C. E. (1976). 'The role of complement
in hydatid disease: in vitro studies.‘ Int. J. Parasitol.
6:25-36.

KABAT, E. A. and MAYER, M. M. (1961). InE. A. Kabat, ed.
Experimental Immunochemistry, 2nd ed., Chapt. 4, Charles C.
Thomas, Springfield, Illinois.

LEID, R. W. and WILLIAMS, J. F. (1974). 'The immunological response
of the rat to infection with Taenia taeniaeformis. II. Character-
ization of reaginic antibody and an allergen associated with the
larval stage.‘ Immunology. 27:209-225.

63

LEPOW, I. H., DIAS DA SILVA, W. and PATRICK, R. A. (1969). In:
Henry A. Movat, ed. Cellular and Humoral Mechanisms in Anaphylaxis
and Allergy. Proceedings of the Third International Symposium
of the Canadian Scoiety for Immunology, S. Karger AG., Basel and
New York, p. 237-252.

MULLER-EBERHARD, H. J. and FJELLSTROM, K. E. (1971). 'Isolation
of the anticomplementary protein from cobra venom and its mode
of action on C3.‘ J. Immunol. 107:1666-1672.

ROCHA E SILVA, M. and GRANA, A. (1945). 'Shock produced in dogs
by hydatid fluid.‘ Am. J. Physiol. 143:306-313.

SCHANTZ, P. M. (1973). 'Homocytotropic antibody to Echinococcus
antigen in sheep with homologous and heterologous larval cestode
infection.’ Am. J. Vet. Res. 34:1179-1181.

TABATABAI, M., BOROOMAND, K., GETTNER, S. and NAZARIAN, I. (1973).
'Respiratory and cardiovascular responses resulting from intravenous
injection of sheep hydatid cyst fluid to dogs.‘ Exp. Parasitol.
34:12-21.

VARELA-DIAZ, V. M. and COLTORTI, E. A. (1974). 'Limitaciones de
la intradermorreaccion de Casoni en el inmunodiagnostico de la

hidatidosis humana.‘ Boletin de la Oficina Sanitaria Panamericana.
76:400-405.

VARELA-DIAZ, V. M., WILLIAMS, J. F., COLTORTI, E. A. and WILLIAMS,
C. S. F. (1974). 'Survival of cysts of Echinococcus granulosus
after transplantation into homologous and heterologous hosts.‘
J. Parasitol. 60:608-612.

WILLIAMS, J. F. and PEREZ-ESANDI, M. V. (1971). 'Reaginic antibodies
in dogs infected with Echinococcus granulosus.‘ Immunology
20:451-455.

INTERACTION BETWEEN TAENIA TAENIAEFORMIS

AND THE COMPLEMENT SYSTEM

B. HAMMERBERG AND J. F. WILLIAMS

Department of Microbiology and Public Health
Michigan State University

East Lansing, Michigan 48824

64
SUMMARY. Factors present in the cystic bladder fluid of metacestodes
of Taenia taeniaeformis and released by these parasites maintained
in vitro were shown to interact with the complement system in vitro
and in vivo. This interaction resulted in the depletion of hemolytic
complement in normal sera of several species, the generation of
anaphylatoxin-like activity in vitro, the conversion of C3, and the
production of vascular permeability changes in vivo. The substances
appeared to initiate complement fixation non-immunologically via
both the alternative and classical pathways. Intravenous adminis-
tration of parasite factors in rats led to profound depression of
circulating complement levels and the rats did not become refractory
to this effect after repeated dosing over a 3 week period. Circulating
levels of hemolytic complement were not altered in infected rats over
the first 8 weeks of infection. Complement-dependent inflammatory
responses in the skin of rats were inhibited in animals given
intravenous doses of parasite factors. The possibility is raised
that local consumption of complement around the metacestode in vivo
could contribute to its successful evasion of inflammation and

immune rejection during infection.

INTRODUCTION
It has been known for many years that infection of the rat
with Taenia taeniaefbrmis results in the development of a specific
acquired resistance to challenge infection which is antibody-mediated
(1,2). In recent studies it has been shown that protective antibodies
produced during the first several weeks of infection are exclusively
in the 1962a immunoglobulin subclass (3,4) and that depletion of

complement in challenged recipients of protective antibodies

65

interferes with the manifestation of resistance (5). Nevertheless,
parasites derived from primary exposure to infection continue to
survive in the tissues of highly immune rats, and this ability

to evade immunologic attack is rapidly acquired during larval
development (5,6).

The continued survival of the parasite would appear to depend,
at least in part, on successful avoidance of complement-mediated
effector mechanisms. In preliminary studies we found that the
fluid surrounding larvae of T. taeniaefbrmis in situ in hepatic
cysts was able to fix complement in normal serum from rats or guinea
pigs (7). We have further investigated this phenomenon and report
here on the interaction of larval factors with the complement
system. These interactions result in; consumption of complement
via both the alternate and classical pathways; conversion of C3
and the generation of anaphylatoxin in vitro; the depression of
circulating hemolytic complement activity, and alterations in
vascular permeability in vivo. In an accompanying paper we describe
the physico-chemical characteristics of substances extracted from

larvae which are responsible for these effects.

MATERIALS AND METHODS

Reagents

Carbowax PEG 20,000 (Union Carbide, Cleveland, Ohio) was
purchased from Fisher Scientific Company. Bovine serum albumin
(BSA), bradykinin triacetate, histamine dihydrochloride, and cypro-
heptadine, were obtained from Sigma Chemical Company. Mepyramine

maleate was a gift from Merck, Sharp and Dohme Research Laboratory

66

(Rahway, New Jersey). Diphenhydramine hydrochloride (Benadryl)
was purchased from Parke, Davis and Company (Detroit, Michigan).
Cobra venom from Naja haje was purchased from Sigma Chemical
Company and the active anticomplementary cobra venom factor (CoF)
was purified chromatographically by the method described by Ballow
and Cochrane (8).
Sheep red blood cells (SRBC) were collected in sterile Alsever's
solution and stored at 4°C before use. Antiserum to washed SRBC
was prepared in rats by injecting 8 X 106 cells intraperitoneally
into mature 200 9 rats and collecting serum 8 days later. A specific
antiserum against purified human complement component C3 was prepared
in sheep. C3 was generously supplied by Dr. R. A. Patrick. This
reagent produced a single band in double diffusion and immunoelectro-
phoresis tests with purified C3 and with normal human serum (NHS),
and prior incubation of NHS with zymosan resulted in conversion

of the band to a more anodally migrating form.

Sources of Parasite Materials

 

Female rats (Spb [SD] BR) 28 days old were obtained from
Spartan Research Animals, Haslett, MI, and each was dosed orally
with 500 eggs of Taenia taeniaeformis. Eggs were liberated by
dissection from gravid tapeworm segments collected from the feces
of experimentally infected cats, washed in saline and stored at
4°C in distilled water containing penicillin (1000 i.u/ml),
streptomycin (100 ug/ml) and fungizone (2.5 pg/ml). Three to
five months later the rats were killed with CO2 vapor and parasites

were dissected from the hepatic cysts (Fig. 1). These larvae were

washed 10 times by sedimentation in 10 volumes of distilled water,

67
and 3 times in sterile 0.15M NaCl before transfer to sterile flasks
containing tissue culture medium. Fifty larvae were incubated
with 200 m1 of Hanks' BME at 37°C and the supernatant fluid collected
after 24 hrs. By following this procedure bacterial contamination
of the medium was avoided in almost all cultures. Those which
became contaminated were discarded. Parasites maintained in this
manner in vitro retained motility, gross morphologic appearance
and infectivity for cats. Supernatant fluids were concentrated
lOO-fold by dialysis against Carbowax prior to dialysis against
distilled water for 48 hr at 4°C. This preparation was termed
in vitro products (IVP).

Bladder fluids from the cystic parasites were collected by
needle puncture of larvae in infected livers at 3-5 months of in-
fection and concentrated 3-fold by Carbowax treatment before dialysis
against distilled water for 48 hr. This preparation was termed
cyst-fluid (CYF). Bladder fluids were similarily collected from
parasites in the livers of rats infected for 20 days. In some
experiments larvae freed from the liver by dissection were incubated
directly in normal serum samples and the remaining complement

activity in the serum assayed after 30 min at 37°C.

Absorption of Host Serum Proteins from Parasite Material

Antisera against normal rat serum (NRS) or rat immunoglobulin
were polymerized by glutaraldehyde treatment, according to the
method of Avrameas and Ternynck (9). Polymerized antisera were
used to absorb out rat serum proteins which were present in cyst
fluid. The effectiveness of absorption was determined by double

diffusion in gel tests.

68

Assay for Hemolytic Complement

 

Hemolytic complement titers were measured in CH50 units
following a modification of the procedure of Kabat and Mayer (10).
Sheep erythrocytes (SRBC) were sensitized with rat anti-SRBC sera.

3 X 108 sensitized cells (EA) in 0.5 ml of veronal buffered saline
containing 1.5 X 10'4M Ca++ and 5 X 10"4 M Mg++, pH 7.4, ionic strength
0.15, were added to 0.5 m1 of the complement source. After incubation
at 37°C for 1 hr, 2 m1 of buffer were added to each tube and the
absorbance at 541 nm recorded after centrifugation at 1000 g for

10 min. To assay for complement-fixing activity the inhibitor in
distilled water was incubated with equal volumes of either normal
guinea pig serum (GPS) or NHS for 60 min at 37°C, and the mixture
assayed for hemolytic complement as described above. Comparison

with the distilled water control CHSO level provided a percent
inhibition value. A modification of the above procedure, described
by Sakamoto and Nishioka (11), was used when measuring rat hemolytic
complement titers; in this modification dilutions of rat serum plus

EA were incubated at 22°C for 2 hr instead of 37°C for 1 hr.

Quantitation of C3 Activation Via Alteranate vs. Classical Pathway

 

EAC I42 were prepared by the method described by Leon (12)
in which 90 m1 of EA at 3.0 X 108 cells/ml were incubated with 22.5 ml
of a 1:10 dilution of NHS previously incubated with zymosan (Sigma)
at a concentration of 15 mg/ml for 60 min at 37°C. The incubation
time of EA with zymosan-treated serum was variable depending upon
optimal EACI42 formation. These cells were used immediately after
being washed in cold triethanolamine-buffered saline (TBS), pH 7.5

containing 1.5 x 10'4 M ea++ and 5 x 10-4 M Mg++. The EAC 142

69

Figure 1. Liver of rat infected with cystic larvae of
Taenia taeniaefbrmis (above). Larvae dissected from within the host
capsule (below). The fluid filled bladder is indicated by the arrow.

 

 

71

were lysed in dilutions of guinea pig serum (GPS) containing 0.04 M
EDTA.

Equal volumes of CYF or IVP or distilled water were incubated
at 37°C for 1 hr with GPS containing no chelators, 0.04 M EDTA,

or 0.04 M EGTA plus 0.04 M MgCl Serial dilutions in 2.5 m1

2.
volumes of TBS plus 0.04 M EDTA of the incubated solution were combined

8 EAC 142/m1 before incubation again at

with 0.5 ml Of 5.0 X 10
37°C for 1 hr. Hemolysis was quantitated by absorbance at 541 nm

after centrifugation. *CHSO units per ml were calculated according

to methods described by Kabat and Mayer (10).

Immunoelectrophoretic Analysis

 

Immunoelectrophoresis was employed to detect conversion

of human C3 to a faster migrating species. The test was conducted
using microscope slides coated with 2% agarose in a veronal buffer
pH 8.6 and current applied for 2 1/2 hrs at 15 mamp/slide. Troughs
were filled with monospecific anti-human C3. Positive conversion
slides were obtained by incubation of human serum with an equal
volume of zymosan (1 mg/ml) at 37°C for 1 hr before electrophoresis.
The influence of Ca++ and Mg++ chelation was determined by prior
treatment of electrophoresed samples with either 0.01 M EDTA or

0.01 M EGTA plus 0.05 M MgClz.

Assay for Anaphylatoxin Activity

 

Normal rat serum samples incubated with an equal volume of
CYF or IVP at 37°C for 1 hr were assayed for the presence of
anaphylatoxin using the bioassay procedure described by Dias Da

Silva et al (13). This involved measurement of the deflection in

72
millimeters of a balanced lever arm attached to a 1 cm segment of
guinea pig ileum suspended in a bath of Tyrode's solution at 37°C.
Deflections were registered after additions to the bath of 50 ng
of histamine, 150 ng bradykinin, or 0.05 ml of NRS incubated with
either CYF, IVP or noble agar. The criterion fortfluaproduction of
anaphylatoxin in NRS was the generation of tachyphylaxis and cross
densensitization of the ileum with repeated application of NSR-agar,
NRS-CYF or NRS-IVP samples. Ileum preparations were treated with
50 ngm of the antihistamine (mepyramine maleate) in order to
distinguish between contractile effects mediated by histamine and

those due to bradykinin.

Assay for Localized Vascular Permeability Changes

 

Four groups of four rats were shaved over the back and each
rat received intradermal injections of 0.05 ml of a solution containing
1 ug/ml histamine, CYF, IVP, or PBS followed immediately by intravenous
inoculation of 0.5 m1 of a 1% solution of Coomassie Brilliant blue.
One hr before skin testing three of the groups received one of the
following antihistamines: diphenylhydramine HCl at 20 mg/kg, mepyramine
maleate at 20 mg/kg, or cyproheptadine at 20 mg/kg. These dosages
of antihistamines were chosen since they prevented any detectable
bluing at histamine injection sites. The influence of systemic
complement depletion on the skin bluing reactivity of each preparation
was determined by the intravenous administration of chromatographically
purified CoF prior to skin sensitization. Total hemolytic complement
levels were determined in rats prepared in this way and found to

be consistently less than 5% of normal values.

73

Passive Cutaneous Anaphylaxis

 

Complement-dependent, histamine-mediated passive cutaneous
anaphylaxis (PCA) reactions were generated in normal adult rats
by intradermal injection of serum containing IgG2a antibodies from
rats sensitized to bovine serum albumin (BSA) (14), followed 2 hrs
later by intravenous administration of 0.5 ml of BSA (1 mg/ml)
containing 1% Coomassie Brilliant blue. Diameters of blued areas
in the skin were compared. Solutions to be assayed for complement
depleting activity were given intravenously 30 min prior to BSA
challenge. Positive controls for skin bluing in complement-depleted
rats were established by intradermal administration of 0.05 ml of

solutions of histamine (1 ug/ml).

RESULTS

The addition of CYF or IVP to normal serum of rats, guinea
pigs or humans in the proportions described above consistently
resulted in depression of total hemolytic complement levels (measured
in CHSO units) (Table 1). Preincubation of EA in parasite factors
followed by washing in buffer, resulted in no change in their sus-
ceptibility to complement-mediated lysis. Rabbit antiserum against
whole normal rat serum (anti-NR8) and sheep anti-rat Fab produced
several precipitin bands when examined in double diffusion tests
against CYF, but none against IVP. Absorption of CYF with polymerized
rabbit anti-NR8 removed all detectable activity with these antisera.
However, the capacity of absorbed CYF to deplete complement levels
in normal serum samples was unaffected. Absorbed CYF was used in
the experiments described below. Cyst fluid aspirated in small

amounts from cysts 20 days after infection was found to inhibit

74

TABLE I

Percent depression of total hemolytic complement levels

 

Source of Serum source
parasite material Rat Guinea Pig Human
CYF 70 60 50

IVP 75 7O 6O

 

75

complement to the same extent as cyst fluid from later stages of
development. A single live 2 month-old parasite incubated in 1 m1
of NRS resulted in a decline of total hemolytic complement to less

than 5% of the normal value in 30 mins.

The results of CYF or IVP incubation with guinea pig serum

in the presence of 0.04 M EDTA or 0.04 M EGTA plus 0.04 M MgC12

are shown in Table 2. Complement depletion was reduced by 37%

in the EGTA-MgC12 chelated preparations, but was inhibited almost

totally in EDTA-treated mixtures.
After incubation of CYF or IVP with equal volumes of normal

human serum for 1 hr at 37°C, the mixtures were subjected to

irmnunoelectrophoretic analysis using sheep anti-human C3. Control

samples of serum were incubated with either buffer or activated

zymosan. The results are shown in Fig. 2. Conversion of C3 was

evident in the NHS-parasite factor mixtures, but no detectable change

occurred in the control serum. Conversion was seen in the zymosan-

serum mixture. When EDTA was added to each incubation mixture at
a concentration of 0.01 M, no conversion was observed; however,

0.01 M EGTA plus 0.05 M MgC12 did not block completely the conversion
of C3 by parasite factors, and had no effect on zymosan conversion.

Incubation of CYF or IVP with NRS at 37°C for 60 min resulted

in the generation of smooth muscle-contracting activity. The addition

of 0.05 ml of this mixture to the isolated ileum preparation produced

a greater degree of contraction than that obtained with 50 ng of hista-

mine (Fig. 3) . Contractile activity was also generated by the incuba-
tion of NRS with agar. After repeated application of the NRS-agar

mixture the ileum became refractory to NRS-CYF or NRS-IVP, but

76

TABLE II

Effect of differential cation chelation on C3 consumption by
products of T. taeniaefbrmis

 

 

Complement source Percent consumption
GPS + IVP 75
GPS + IVP + EDTA 2

GPS + IVP + EGTA + Mg++ 47

 

77

 

Figure 2. Immunoelectrophoretic analysis of human serum
following incubation with either CYF (top) or zymosan (bottom) -
Normal serum was used as a control. The troughs contained sheeP
anti-human C3.

 

 

 

 

Figure 2

79

Figure 3. Smooth muscle contraction in response to sequent-'1?"l
stimulation. Vertical bars represent height of contraction (mm)
registered by guinea pig ileum strip suspended in bath to which
were added histamine (Stippled bar), CYF plus normal rat serum
(dark stripe) or agar plus normal rat serum (light stripe).

 

 

 

 

80

m musmflm

 

u>0

 

m>0+wmz

 

 

m>0+wmz

 

 

m>0+wmz

 

 

m<o<+mmz________________________________________________ ”:6
+wmz

 

 

 

 

 

81

remained responsive to the addition of 50 ng histamine. In the

reciprocal experiment tachyphylaxis was produced by the repeated

application of NRS-CYF and the preparation was also refractory

to NRS-agar but responsive to histamine: NRS, CYF or IVP alone

did not produce any contraction. Mepyraminized ileum preparations

were responsive only to bradykinin (150 ng) .
When CYF or IVP preparations were inoculated intradermally

into the backs of normal rats, intense local bluing occurred after

intravenous administration of Coomassie Brilliant blue dye. Reactions

were most intense by 10 min and reached a diameter of 8 mm, comparable

to that achieved with 50 ng histamine. Control inoculations of

PBS or distilled water produced only minor bluing at the point of

needle trauma. Pretreatment of rats with antihistamine completely

abolished the reaction at sites of histamine inoculation but did

not completely ablate skin bluing due to CYF. Intravenous doses

of CoF resulted in a high degree of inhibition, but skin bluing was

still slightly visible at sites inoculated with CYF.
Intradermal inoculations of CoF produced intense bluing in
the skin of normal rats which had been given dye intravenously.

However, when rats were given 0.5 m1 of CYF or IVP along with the

dye, skin reactivity to CoF was abolished. When rats were prepared

for the complement-dependent PCA reaction by the intradermal inocula-
tion of serum containing IgGZa-antibodies to BSA, intense bluing

occurred at sensitized sites after intravenous administration

of BSA plus dye 2 hrs later. When CYF or IVP (0.5 ml) was given

along with the BSA-dye mixture the PCA reaction was completely

abolished. However, these rats remained responsive to intradermal

82
inoculation of 50 ng histamine.

In an attempt to determine if large numbers of parasites
developing in the liver of a rat could depress circulating complement,
C350 levels were measured serially during the course of an infection.
Six 28 day old rats were bled from the orbital plexus prior to
infection with 1000 eggs of T. taeniaefbrmis and then bled at 7 day
intervals thereafter for 60 days. Six uninfected rats of the same
age and from the same litter were also bled. The results are shown
in Fig. 4. Both groups showed fluctuating levels with an upward
trend in circulating hemolytic complement levels over the period
of study but no differences were apparent. More than 100 parasites
were present in the liver of each infected rat at necropsy.

Intravenous administration of either CYF or IVP to normal
rats resulted in a fall in circulating hemolytic complement levels.
One half ml of a preparation of CYF which was able to deplete hemolytic
complement in vitro by 60% caused a reduction of circulating complement
in Vivo to 15-20% of normal (pre-inoculation) level within 30 min.
Restoration of normal levels occurred in 24 hr. When rats were given
the same dose intraperitoneally the decline was much less pronounced
and levels fell by only 50%, returning to normal in about 8 hr.

In order to determine whether rats became refractory to the com-
plement-depleting effects of CYF, 5 rats were given intraperitoneal
inoculations of 0.5 ml CYF every 8 hrs for 6 days. On the seventh
day intravenous administration of 0.5 ml CYF produced a decline in
CHSO levels to approximately 50% of normal values. Injections of
CYF were continued.at weekly intervals for 3 weeks in these same

rats and after the third dose hemolytic complement was shown to

Figure 4.

83

Serial determinations of serum complement (CHSO)

levels of rats dosed with 1000 eggs of Taenia taeniaeformis

at 28 days of age.

Control rats (0-0) were uninfected littermates.

 

 

o-o Infected
o—oCOfltl‘Ol

 

 

7O

(lw/OSHO) lNawa'Idwoo

b(D

I-ID

 

WEEKS

Figure 4

85
have fallen by 50% of the pre-inoculation level in 30 minutes.
Serum samples from these rats were tested in double diffusion against

CYF but no precipitin bands were detectable.

86

DISCUSSION

These experiments indicate that factors present in the cyst
fluid of T. taeniaeformis as early as 20 days after infection, and
released by live worms maintained in vitro, are able to interact
in a non-immunologic manner with the complement system. Consumption
of hemolytic complement occurred in serum of normal rats, the natural
host, and in normal guinea pig and human sera. The reaction did not
appear to be dependent on the presence of immune complexes in the
parasite cyst fluid, since immuno-absorption effectively removed
detectable immunoglobulins but did not affect the complement depleting
activity of this material, and immunoglobulins were not detectable
in the in vitro culture supernatants. Consumption of complement
was inhibited by EDTA but was only partially (37%) inhibited by
selective chelation of Ca++ suggesting that the reaction could
proceed to some extent via the alternate pathway (15,16). Similarily,
the conversion of C3 to an electrophoretically faster migrating
species was only partially inhibited under conditions which permit
the activation of complement via the alternate pathway (17).

Smooth muscle-contracting substances generated by the incubation
of parasite materials with NRS appeared to be analogous to anaphyla-
toxins since reciprocal cross-tachyphylaxis occurred with NRS-agar
preparations, the activity was inhibited by antihistamine, and respon-
siveness to bradykinin was unaffected. However, in vivo vascular
permeability changes in rats differed from those due to anaphylatoxins
in that substantial but not complete inhibition was seen in both
antihistamine-treated and in complement-depleted rats. This finding

suggests that parasite materials may influence permeability through

87

mechanisms other than those involving the complement system. Our
recent finding that substances present in cyst fluid interact
with the intrinsic coagulation pathway may be of significance

in this regard.1

Parasite materials were evidently able to deplete complement
levels in vivo resulting in profound reduction in CHSO titers and
in the ablation of skin-bluing produced by CoF or by complement-
dependent antigen-antibody-mediated cutaneous inflammation. The
latter finding is particularly important in view of the observation
of Leid and Williams (18) that IgGZa antibodies to T. taeniaeformis
were ineffective in PCA reactions. In their experiments rats
sensitized intradermally with 1962a failed to show skin-bluing upon
intravenous challenge with blue dye plus a crude antigenic extract
of T. taeniaeformis, whereas IgE-mediated reactions were consistently
provoked with this extract. They suggested that IgG2a antibodies
produced in response to infection might differ from those classically
stimulated by parenteral inoculation of antigens. In retrospect,
however, it seems likely that the crude extract inoculations were
causing rapid depletion of complement and hence inhibiting the
IgGZa-mediated PCA, but permitting the complement-independent IgE-
mediated PCA reactions (14) to proceed normally.

The pattern of in vivo complement-depletion produced by
inoculation of parasite materials differed from that observed with
CoF treatment. Repeated exposure did not appear to lead to a
refractory state, comparable to that which occurs in 5-6 days when

rats are given multiple doses of CoF (5). The onset of refractoriness

Footnote 1. Manuscript submitted for publication.

88

is generally attributed to immune elimination following recognition

of antigenic determinants in the active venom protein (19) . Rats

which had received 20 inoculations of CYF over a 4 week period still
suffered acute, though less profound, reductions in circulating

complement shortly after treatment, and no precipitating antibodies

were detected in their sera. This suggests that the complement

interacting substances have very limited antigenicity in the rat.
It may be that the recently reported anticomplementary activity
of pentosan-poly-sulfoester in humans is more closely analogous

to the activity of larval substances (20).

In spite of the efficacy of the parasite materials in depleting
complement in vivo after inoculation, there was no evidence of lowered

levels in the serum of rats during the first two months of infection.

This could be attributable to; the occurrence of little or no release

of complement-interacting materials by the parasite in vivo; effects
of released factors being exerted only at the local level around the
cyst; or systemic effects on circulating complement being counteracted

by homeostatic mechanisms which tend to compensate and restore normal

levels. In view of the fact that hemolytic complement is not detect-

able in the plasma-protein rich fluid surrounding the parasite
in situ (7) we believe that release probably does occur in vivo,
but possibly at a rate insufficient to alter systemic levels.
Experimental quantitation of release in vivo may be achieved if
specific markers can be incorporated into the active molecules,
once they have been characterized.2

The significance, if any, of these complement-fixing substances

Footnote 2. Manuscript submitted for publication.

 

89
in the host-parasite relationship cannot be assessed at this time.
The susceptibility of taeniid metacestodes to antibody-mediated attack
in the presence of heat-labile serum factors(5) , and the finding
that complement depletion in vivo interferes with the efficacy
of passive immunization (5) , indicate that prolonged survival

in the tissues might involve interference with effector mechanisms

which depend on complement. Indeed, under some circumstances com-

plement alone appears to be able to have a deleterious effect on

taeniid parasites (21) . local consumption of complement in the

soluble phase by factors released by these parasites might provide

a defensive mechanism for evasion of either non-specific or specific

attack. Similar mechanisms have been proposed for the survival of

bacteria. However, such a reaction would be expected to lead to

the generation of anaphylatoxic activity which, in turn, would

result in cellular infiltration and local vascular changes. In

serial studies of the histopathologic response during primary
infection there was a very marked infiltration of eosinophils around

hepatic parasites, which reached peak levels during the fifth week,

but then subsided (22). After several months of infection, some

eosinophils were still present in the connective tissue of the local
host reaction around the worms but there was no evidence of an

acute inflammatory process which one might anticipate if local

complement fixation were taking place. In this study, anaphylatoxic

activity was not detectable in cyst fluid samples applied directly

to the smooth muscle preparation. It is possible that endogenous

enzymatic deactivation of anaphylatoxins may lead to reduced

inflammatory activity, particularly if they are produced but then

9O

deactivated at some distance from the blood vasculature of the

connective tissue capsule. It is also possible that proteolytic

enzymes released by the parasites contribute to degradation of

biologically active complement fragments. There is histochemical

evidence of release of proteases by T. taeniaeformis in situ (23)
and the enzymatic activities of in vitro parasite products could

be examined experimentally to explore this hypothesis.

In recent work we and others (21,24,25) have detected complement

interacting substances in a variety of taeniid cestodes. Whatever

the role of these substances may be during infection, their presence

provides some basis for interpreting several other phenomena associated
with these organisms. In particular the high proportion of normal

people who show skin reactivity to taeniid extracts (26) could be

due to the non-immunologic generation of vascular permeability

factors. Also the anaphylactic-type shock syndrome which can be

provoked in non-infected normal animals by the intravenous admin-

istration of taeniid extracts might be due to systemic effects of

massive complement fixation (27). Identification of the nature

of these factors may therefore be relevant in context of the clinical

diagnosis of taeniiases as well as in basic investigations on host-

parasite relationships and evasion of rejection. Details of the

physico-chemical characterization of these substances in T.

taeniaeformis are presented in an accompanying paper.2

ACKNOWLEDGEMENTS

This work was supported by NIH Grant AI-10842. We are indebted
to Dr. A. J. Musoke for helpful advice in several aspects of this

study .

91

This is Journal Article No. from the Michigan

Experimental Station .

.10.

ll.

12.

92

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globulin classes involved in passive transfer of resistance.

Immunology 27:195.
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Ballow, M., and C. G. Cochrane. 1969. Two anti-complementary
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Avrameas, S., and T. Ternynck. 1969. The cross-linking of
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Jap. J. Exp. Med. 45:183-189.
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Dias Da Silva, W., J. W. Eisele, and I. H. Lepow. 1967.
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126:1027-1048.

Stechschulte, D. J., R. P. Orange, and K. F. Austen.
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of mediating immediate hypersensitivity reactions in the rat,
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Interaction of complex
Effect of calcium

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Immunity 11:273-279.
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Muller-Eberhard, H. J., and K. E. Fjellstrom.
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Leid, R. W., and J. F. Williams. 1974.
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PHYSICO-CHEMICAL CHARACTERIZATION OF COMPLEMENT-INTERACTING

FACTORS FROM TAENIA TAENIAEFORMIS

BRUCE HAMMERBERG AND JEFFREY F. WILLIAMS

Department of Microbiology and Public Health
Michigan State university

East Lansing, Michigan 48824

Skuxrt.Title: Complement interacting factors of T. taeniaeformis

ABSTRACT

Complement-fixing activity was washed from the surface of
metacestodes of Taenia taeniaefbrmis and characterized physico-
chemically using enzymatic digestion, gel-filtration, anion exchange
chromatography, ultracentrifugation and gel electrophoresis. The
results provided evidence of an association between antihemolytic
activity and macromolecules containing carbohydrate, protein, sulphate
and hexosamine, heterogenous in net negative charge but resistant
to proteolysis and B elimination reactions, and free of sialic
and uronic acids. The most active fraction was eluted from DEAE-
cellulose with 0.3 M NaCl in 0.01 M Tris-HCl pH 8.0, and when parasites
were incubated with a source of 35804", radioactivity was incorporated
into molecules eluting under identical conditions. Active chroma-
tographic fractions precipitated with protamine sulphate, suggesting
that they were polyanionic in nature. There was some evidence of
dissociation of the active substance on polyacrylamide gels where
fast-moving sulphate-containing bands were observed which no longer
.reacted with protamine or complement.

The results are consistent with the possibility that the active
substance is a polysulfated proteoglycan. Molecules of this nature
ruave been detected on the surface of a variety of infectious organisms,
arni are known to interact with complement non-immunologically.

Tfimeir location at host-parasite interfaces may have strategic
sirgnificance in evasion of immune rejection and this possibility

jig; discussed in relation to current understanding of immunity to

T . taeniaeformis .

95

96

INTRODUCTION

In a previous report we showed that substances in the cyst
fluid of metacestodes of Taenia taeniaefbrmis and released into
the surrounding medium in vitro could apparently initiate complement
fixation non-immunologically via both the classical and alternate
pathways.l These findings are comparable to results obtained with
certain bacterial lipopolysaccharides (1,2). We have therefore
pursued the physico—chemical characterization of parasite factors
in order to determine if they might be structurally analogous to
the bacterial products, and our results are presented here.

Maximal complement-depleting activity was consistently
associated with a fraction containing a highly sulfated polysaccharide
or proteoglycan. Recently it has been shown that molecules of
this nature may interact with complement in a variety of ways
(3,4,5,6), and their occurrence at bacterial surfaces has been
considered of primary importance in the host-parasite relationship
(7,8). It is suggested their presence at the surface of tissue-
dwelling metacestodes might also be significant in the avoidance

of host rejection mechanisms.

MATERIALS AND METHODS

.Reagents

Bovine serum albumin (BSA), glucosamine, glucose, N-acetyl
Jmeuraminic acid, papain insolubilized on carboxymethylcellulose,
Ichondroitinase ABC, chondro-4-sulfatase, chondro-6-sulfatase,

El-glucuronidase, neuramdnidase, B-galactosidase, and deoxyribonuclease

Footnote 1. Manuscript submitted for publication.

97
were purchased from Sigma Chemical Company, Saint Louis, Missouri.
Ethylenediamine tetra—acetic acid disodium salt (EDTA), polyethylene
glycol, 20,000 (Carbowax), and pepsin were purchased from Fisher
Scientific Company, Fair Lawn, New Jersey. Anthrone reagent and
cetylpyridinium chloride monohydrate were purchased from Aldrich Chem-
ical Company, Inc., Milwaukee, Wisconsin. Trypsin (1:250) was obtained
from Difco Laboratories, Detroit, Michigan. H23SSO4 was obtained from
New England Nuclear, Boston, Massachusetts. Granular (pre-swollen)
anion exchanger, Whatman DE-52, was purchased from Reeve Angel, Clifton,
New Jersey. Sephadex G-200 was purchased from Pharmacia Fine Chemicals,
Inc., Piscataway, New Jersey. Sepharose 6-B was purchased from Bio

Rad Laboratories, Richmond, California.

Preparation of Larval Extracts and in vitro Products

Larvae of Taenia taeniaefbrmis (3-5 months old) were recovered
from the livers of rats as described previously. They were washed
10 times with 10 volumes of sterile 0.15M NaCl and then incubated
in 0.1 M Tris-HCl buffer, pH 8.0, containing 0.02 M EDTA at 37°C
for 30 min. Four volumes of the Tris-HCl-EDTA buffer were used for
(every volume of packed larvae. After incubation, MgC12 was added
tx: the extract solution to make it 0.05 M, before dialysis against
(distilled water and 15-20 fold concentration by Carbowax. Final
exhaustive dialysis against distilled H20 was carried out to remove
aury Carbowax which had entered the dialysis sac. This product will
rue referred to as Tris-EDTA extract (T.E.). The medium in which
liarvae had been maintained at a concentration of 50 larvae/200 ml
for 24 hrs was concentrated 100 fold, and then dialysed against

(mistilled water for 48 hrs; this product will be referred to as

98
in vitro products (IVP).
Radio-labelled IVP was obtained by adding 6 mC of H235804
to a culture flask in which 50 larvae were maintained in 200 ml
of medium for 24 hrs. Labelled material was counted in a Packard

liquid scintillation counter (Packard Instrument Co., Downer's

Grove, Illinois.).

Assa 5

Protein concentrations were determined by the method described
by Lowry et a1 (9) using BSA as a standard. Neutral sugars were
assayed using the Anthrone reagent and glucose as a standard (10).
.Amino sugars were detected by the micro-technique developed by Gatt
and Berman (11) and concentrations were determined by comparison
‘with.a glucosamine standard. Sialic acid was assayed by the technique
(described by Aminoff (12), with a neuraminic acid standard. Uronic
acid.concentrations in unknown samples were measured using the method
of Dische (13).

Labile SO4" determinations were conducted by a modification
(If the method described by Saito et a1 (14). One tenth ml of 0.2 N HCl
was added to 0.4 ml of the sample in an ampoule which was then sealed
and heated at 100°C for 2.5 hrs. The seal was broken and 0.1 m1 of
0.22 N NaOH was added, followed by 2.0 m1 of 0.5% cetylpyridinium
ii: 0.3 N HCl to precipitate polysaccharides. After 10 min at 37°C
2.11 ml of BaC12 gel in 0.3 N HCl was added to 0.9 ml of the supernatant
axui the optical density at 530 nm was measured 10 min later. When
K2804 was used as a standard, the optical density was linearly related

to concentration over the range 5-80 ug/ml.

99
Enzymatic Digestion

For the tryptic digests 1 mg of trypsin 1:250 was added
to each milliter of T.E. containing 10 mg of protein in 0.2 M NaHCO3,
pH 8.0. The mixture was incubated at 37°C for 48 hrs, with 0.1%
sodium azide added to prevent microbial growth. Trypsin was removed
after dialysis by precipitation with 10% trichloracetic acid (TCA).
Peptic digestion was also carried out for 48 hrs at 37°C using
5 mg of pepsin for each milliter of T.E. containing 10 mg of protein
in 0.1 M acetate buffer, pH 4.5. Pepsin was removed by molecular
sieving on Sephadex G-200 or by precipitation with 10% TCA. Papain
(50 mg), insolublilzed on carboxymethyl cellulose, was incubated
for 24 hrs at 37°C with one milliter of T.E., (containing 10 mg/ml
protein) in 0.1 M sodium phosphate buffer, pH 7.0, and 0.1 M l-cysteine
plus 0.002 M EDTA. The papain was removed by centrifugation and
the digest dialysed against distilled H20. In a similar manner,
lprotease from Streptomyces griseus covalently bound to carboxymethyl
cellulose was added to T.E. in 0.1 M Tris-HCl, pH 7.3. Ten mg of
the protease preparation was incubated with 1 milliter of T.E.
containing 2 mg of protein for 24 hrs at 37°C. Protease was removed
Iby centrifugation and the digest dialysed against distilled H20.

One unit of chondroitinase ABC, 1 unit of chondro-4-sulfatase,
<3r 1 unit of chondro-6-sulfatase was used to digest 1 milliter of
{T.E. containing 1 mg of amino sugar. These enzymes in the amounts
Insed and under the conditions of the hemolytic complement assay
111 control mixtures had no apparent effect on CHSO levels in normal
gjuinea-pig serum. Therefore, they were not removed prior to assaying

ffiar residual T.E. activity in the hemolytic assay. For this same

100
reason the antihemolytic activity in 1 milliter of T.E. containing
2 mg of neutral sugar was assayed in the presence of B-glucuronidase,
neuraminidase, B-galactosidase, or deoxyribonuclease, after incubation
with these enzymes. Bacterial B-glucuronidase (100 Sigma units of
Type II) was incubated with l milliter of T.E. in phosphate buffer,
pH 6.8, for 24 hrs at 37°C. One unit of neuraminidase (Type VE),
purified from Clostridium perfringens, was incubated with one milliter
of T.E. in 0.1 M acetate buffer, pH 5.0, for 24 hrs at 37°C. Eight
units of deoxyribonuclease II from bovine spleen were incubated

with one milliter of T.E. in 0.1 M acetate buffer, pH 4.6 for 16 hrs

at 37°C.

Complement Assays

The procedures for assay of anaphylatoxin and total hemolytic

complement levels were described previouslyl.

Column Chromatography

Anion-exchange chromatography was carried out using DE-52,
equilibrated with 0.01 M Tris-HCl, pH 8.0. Stepwise elution of
fractions was accomplished with NaCl at concentrations from 0.1 M
to 2 M (15).

Molecular sieving using either Sephadex G-200 or Sepharose
(i-B was conducted in columns 100 cm x 2.5 cm and in 0.1 M Tris-HCl
lyuffer, pH 8.0 plus 0.1% sodium azide. In one instance the G-200

Sephadex column was equilibrated with 2 M NaCl in the Tris buffer.

Protamine Precipitation in Agrose Gel

Acid-cleaned microscope slides were coated with 3 ml of 1.5%

agarose in veronal buffer pH 7.4. Wells (2 mm) were punched and

101
filled with solutions of protamine sulfate at 10 mg/ml or with T.E.
preparations or chromotographic fractions. Precipitin bands formed

overnight at 22°C.

Polyacrylamide Gel Disc Electrophoresis

Disc electrophoresis was performed by the methods of Ornstein
and Davis (16). A 5% running gel was prepared in glass tubes
(0.5 cm x 10 cm), in Tri-HCl buffer, pH 8.8. A spacer gel of 4%
cyanogum gel in Tris-HCl buffer pH 7.9 was layered on top to a depth
of 1 cm, and 20 ul of the sample in 5% sucrose was added immediately
before electrophoresis. The reservoir buffer was Tris-glycine,
pH 8.8, and a current of 2 mamp per tube was applied for 3 hr.

After electrophoresis, gels were stained with either 0.1%
Amido Black for proteins or with 1% Toludine blue for acid mucopoly-
saccharide, and destained in 2% acetic acid. Gel slices (2 mm)
were prepared after electrophoresis of the 35804" labelled sample,

and the radioactivity in each segment counted.

RESULTS

Effect of Enzymatic Digestions and Chemical Hydrolysis on T.E. Activity

T.E. preparations were active in hemolytic complement inhibition
and anaphylatoxin generation tests when assayed as described previously
for parasite cyst fluidl. When T.E. was made 10% TCA at 4°C a
precipitate formed which was removed by centrifugation at 10,000 g
for 30 min. After neutralization with NaOH and dialysis overnight,
the supernatant contained approximately 80% of the original antihemolytic
activity.

Incubation of T.E. with pepsin, trypsin, papain, or protease of

102

Streptomyces griseus origin under optimal conditions of temperature
and pH did not reduce its activity. However, each digestion resulted
in a reduction in protein content from 1/3 to 1/4 of the original
level. Similarly, incubations of T.E. in chondroitinase ABC,
glucuronidase, B-galactosidase, chondrosulfatases 4 or 6,
neuraminidase, or deoxyribonuclease were carried out with no
detectable loss of antihemolytic activity.

Alkaline hydrolysis with 0.5 M NaOH at 60°C for 16 hrs had
no effect on T.E., but acid hydrolysis with 0.1 N HCl at 60°C for

5 hrs eliminated 85% of the antihemolytic activity (Fig. 1).

Molecular Sieving and Preparative Ultracentrifugation Studies

The elution profiles of T.E. and IVP, in Sepharose 6 B columns
are shown in Figure 2. The majority of material detectable as protein
or neutral sugar was present in the void volume. The void volume
peak also contained most of the antihemolytic activity. When either
T.E. or IVP were subjected to ultracentrifugation at 106 g for 5 hrs
activity was concentrated in the sedimented material with lesser
amounts in the supernatant. In the presence of 2 M NaCl, most of
the T.E. activity was still excluded from G-200 Sephadex resin, with
only a small amount retarded in its elution (Fig. 3).

When T.E. was digested with pepsin for 48 hrs most of the
activity still eluted from Sepharose 6-B in the void volume (Fig. 4).
This elution pattern was not changed when the pepsin-digested T.E.

‘was treated with 1 M NaOH at 60°C for 1 hr, (Fig. 4).

.Anion Exchange Chromatography

T.E. was applied to a column of DEAE cellulose in 0.01 M

103

Figure 1. Effect of acid hydrolysis of T.E. on inhibitory
activity in a complement-mediated hemolytic assay. T.E. was incubated
with 0.1 N HCl at 60°C, and activity was assayed in samples withdrawn
hourly.

104

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Figure 2. Protein (OD 280 nm) and neutral sugar (OD 620 nm)
elution profiles of IVP (—-) and T.E. (---) from a Sepharose 6-B
column equilibrated with 0.1 M Tris-HCl buffer pH 8.0. The IVP
sample contained 4 mg of protein and 2 mg of neutral sugar. The
T.E. sample contained 12 mg of protein and 20 mg of neutral sugar.

106

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Figure 3. Protein (OD 280 nm) and neutral sugar (OD 620 nm)
elution profile of T.E. from a Sephadex G—200 column equilibrated
with 2 M NaCl in 0.1 M Tris-HCl, pH 8.0. The T.E. sample contained
10 mg of protein and 12 mg of neutral sugar.

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Figure 4. Protein (OD 280 nm) and neutral sugar (OD 620 nm)
elution profile of pepsin-digested (——) and pepsin-digested plus
NaOH-treated (---) T.E. from a Sepharose 6 B column equilibrated
with 0.1 M Tris-HCl buffer, pH 8.0. Prior to treatment the sample
contained 8 mg of protein and 11 mg of neutral sugar. Protein
elution in the NaOH-treated sample was identical, except that there
was no peak corresponding to pepsin (tubes 85-120).

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Tris-HCl, pH 8.0, and subsequently eluted with stepwise additions

of increasing concentrations of NaCl, (Fig. 5) . The fraction eluting

with 0.3 M NaCl had the highest activity in the hemolytic complement
assay and contained the highest concentrations of amino—sugars and

sulfate ions. Neither the T.E. applied to the column nor any of

the concentrated pooled fractions eluted from the DEAE contained any

detectable sialic acid or uronic acid. T.E. was then digested with

pepsin and treated with 10% TCA to remove the pepsin and other proteins
before chromatography on DEAE. The elution profile was not appreciably

different from undigested TE, nor were there any marked alterations

in the quantitative analysis of each fraction (Fig. 5).

In order to verify that the sulfate-containing material in
the 0.3 M NaCl peak was of parasite origin, larvae were incubated

with H23SSO4 for 24 hrs and the non-dialysable labelled material

produced was chromatographed on DEAE as above, (Fig. 6). Counts of

355 were highest in the 0.3 M NaCl fraction, coinciding again with

the peak of antihemolytic activity.

Since the elution pattern on DEAE was not affected by proteolytic
digestion or TCA precipitation, an attempt was made to assay for the

effects of alkaline hydrolysis on the elution of the 0.3 M NaCl

fraction. Glycoproteins with glycosidic linkages between sugar

moieties and amino acids are susceptible to B-elimination reactions

in the presence of 0.5 M NaOH for 24 hrs at 22°C (17). T.E. was

therefore digested with pepsin and then treated with 10% TCA before

DEAE chromatography. The 0.3 M NaCl peak was then subjected to

alkaline hydrolysis. The total sugar content of the fraction was

reduced but the protein content and antihemolytic activity were not

112

Figure 5. Comparison of the protein (OD 280 nm) and neutral
sugar (OD 620 nm) elution profiles of T.E. before (A) and after (B)
digestion with pepsin. Elution of the sample from DEAE cellulose
was accomplished by stepwise addition of increasing concentrations
of NaCl. The sample in A contained 6.0 mg of protein and 10 mg of
neutral sugar. The sample in B after pepsin digestion and treatment
with 10% TCA contained 2.5 mg of protein and 7.0 mg of neutral sugar.
Pooled fractions were concentrated to 3 m1. ND indicates "none
detectable."

113

 

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114

Figure 6. Relationship of protein (OD 280 nm) and neutral
sugar (OD 620 nm) to cpm from 35504" and complement-inhibitory
activity in fractions of IVP eluted from DEAE cellulose. The IVP
sample contained 2 mg of protein, 1 mg of neutral sugar and 4,000 cpm
of 35804". Pooled fractions were concentrated to one m1.

115

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116
changed. Antihemolytic activity and the majority of the non-dialysable
protein and neutral sugar eluted in 0.3 M NaCl fraction after

rechromatrography on DEAE.

Protamine Precipitation

 

When T.E. and fractions of T.E. from DEAE chromatography were
allowed to diffuse in agarose gels against protamine, distinct
precipitin bands formed. Only T.E. and the 0.2 M and 0.3 M NaCl
eluates formed bands, and the 0.3 M NaCl fraction formed a more
dense band closer to the protamine well (Fig. 7). These two
chromatographic fractions still precipitated with protamine in double
diffusion gels after they had been treated with 0.5 M NaOH at 60°C

for 12 hrs.

Polyacrylamide Gel Disc Electrophoresis of T.E. Extracts

 

After polyacrylamide gel disc electrophoresis of T.E.,
T.E. digested by pepsin, or T.E. incubated with l M NaOH (60 min at
60°C) all gels showed the same number and location of bands detectable
with Amido Black or toluidine blue, (Fig. 8). Heavy protein-staining
bands occurred in all samples at the origin of the gel and at the
level of the tracking dye (bromcresol blue), and a lightly-staining
diffuse area was present in the upper 2 cm of the running gel.
Bands staining with toluidine blue appeared at the origin of the gel,
at the tracking dye site and at two areas beyond the tracking dye.
Again a lightly stained area was present in the upper 2 cm. When
IVP labelled with 35504" was subjected to electrophoresis, counts
were concentrated at the level of the tracking dye (Fig. 9).

Protamine—precipitating activity eluted from the gel segments

117

Precipitation bands formed when protamine sulfate

Figure 7.
(10 mg/ml), in the center well (P), initial wash (W), 0.1 M, 0.2 M,
0.3 M, 0.4 M, and 2 M NaCl eluates from DEAE chromatography of T.E.

before (on the right)and after alkaline hydrolysis (on the left)

were allowed to diffuse in agarose gels.

 

119

Figure 8. Polyacrylamide disc gel electrophoresis band pattern
typical of T.E. or T.E. after alkaline hydrolysis or pepsin digestion.
The gel on the left has been stained with Amido Black, the gel
on the right with toludine blue, and the arrow marks the extent

of tracking dye migration.

120

 

 

 

 

 

Figure 8

121

Figure 9. Polyacrylamide gel electrophoresis of IVP labelled
with 35504". Samples were run for 3 hrs at 2 mamp/tube. Tracking
dye was bromocresol blue.

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was restricted to materials in the upper 2 cm, and no precipitation
occurred with the section containing the fast-moving bands. Anti—
hemolytic activity also corresponded with the upper 2 cm section,

and no inhibition was detected in the fast—moving band area.

124
DISCUSS ION

Surface material extracted from fully developed larvae of
T. taeniaefbrmis with 0.02 M EDTA in 0.1 M Tris-HCl interacted with
complement in a similar manner to cyst fluid removed from the
parasite bladdersl. This extraction procedure has been used
traditionally to collect endotoxin from gram-negative bacteria (18),
and its effectiveness provided us a means of standardizing the
preparation of parasite factors for further physico-chemical
characterization. Other substances may be present within cyst
fluid which interact with complement so that this characterization
was necessarily incomplete.

The resistance of antihemolytic reactivity to 10% TCA treatment
and to a variety of proteolytic enzymatic digestions suggested that
the factors responsible were either non-proteinaceous or that the
protein moiety was peculiarly resistant, perhaps by virtue of its
association with other components. The latter seemed likely since
even after proteolysis and 10% TCA treatment the T.E. supernatant
still contained 40% of the original protein content. The insus-
ceptibility of T.E. to several other enzymatic digestions indicated
that antihemolytic activity could not be attributed to molecular
structures closely related to chondroitin sulfate, hyaluronic acid,
8 galactoside-linked saccharides, DNA, or sialic acid. Glycosidic
linkages susceptible to B-elimination by alkaline hydrolysis did
not appear to be involved in the antihemolytic effect since activity
was retained after exposure to 0.5 M NaOH at 22°C for 24 hrs.
However, there was a striking reduction in antihemolytic activity

following hydrolysis in 0.1 N HCl at 60°C. This observation was

125
consistent with the possible involvement of sulphate groups linked
to the nitrogen of amino-sugars, since these bonds are known to be
disrupted by mild acid hydrolysis (19).

Preparative ultracentrifugation and gel filtration of T.E.
before and after digestion with pepsin or alkaline hydrolysis
indicated that the majority of the active material had retained
a macromolecular structure. The fact that this remained so after
alkaline hydrolysis may be indicative of bonding between polysaccharide
side chains (20). Anion exchange chromatography of T.E. and 35804"
labeled IVP demonstrated that the antihemolytic activity was hetero-
geneous with respect to net molecular charge, as were molecules

containing 504 and hexosamine. The radioactivity detectable in
IVP after incubation of larvae with H23SSO4 demonstrated that the
804'" containing materials were of parasite origin and not host-
derived contaminants. There was a marked association between anti-
hemolytic activity, 804'- level and the 0.3 M NaCl eluate from DEAE.
The activity was pepsin-resistant and insusceptible to alkaline
hydrolysis, and the net molecular charge did not seem to be affected
by these treatments because when rechromatographed the fraction
eluted under identical conditions, even though the total neutral
sugar content had been reduced considerably by hydrolysis.

In sum these observations provide evidence of an association
of the anticomplementary activity with macromolecules containing
carbohydrate, protein, sulphate and hexosamine, heterogenous in
net molecular charge but resistant to proteolysis and B-elimination

reactions and free of sialic and uronic acids. The fact that

antihemolytic chromatographic fractions, rich in sulphate, precipitated

126

with protamine suggested that they may indeed be polyanionic, since
protamine is a polycationic protein capable of binding polyanionic
structures (4). In preliminary experiments (unpublished observations)
we found that prior incubation of T.E. with protamine decreased
antihemolytic activity, and similar results have been reported by
Rent et a1 (5) with protamine-heparin mixtures. These conclusions
appear to be at odds with the results of polyacrylamide disc gel
electrophoresis where most of the protein-staining material and
sulfate groups migrated rapidly, suggesting that they were not part
of a large molecule. This happened whether the T.E. was used
untreated, or after protease digestion or alkaline hydrolysis.
The protein-staining band at the tracking dye did not correspond
exactly with the toludine blue-stained, sulfate-containing material.
However, sections of the gel containing fast-moving bands showed
no antihemolytic activity and did not contain protamine-precipitating
materials. Both these activities were restricted to slow moving
molecules in the initial part of the running gel. Possibly non-
covalent bonds (eg. electrostatic) were maintaining a polymeric
macromolecular structure which was stable at pH 8.0 (21), yet
dissociable under conditions of electrophoresis.

On gas-liquid chromatographic analysis of the most active
DEAE fraction (0.3 M NaCl) only two neutral sugars were detected;
galactose and glucose were present in a molar ratio of 5:1, and
a hexosamine, probably glucosamine, was present in amounts equimolar

2

to galactose .

Our physico-chemical evidence suggesting that anticomplementary

Footnote 2. Manuscript submitted for publication.

127

activity is associated with a sulfated polysaccharide or proteoglycan,
is also consistent with the fact that complement interaction similar
to that described for T.E., IVP, and CYF1 can occur with polysulfated
molecules such as heparin (3). Also important in relation to host
inflammatory reactions is the recently described activity of certain
polyanions in enhancing the function of Cl-inhibitor (Cl-INH) (6).
We have not determined if substances of parasite origin are able
to influence Cl-esterase or Hageman factor activity by Cl-INH
enhancement, but we have recently found that T.E. will inhibit the
intrinsic pathway of co-agulationz. These results are particularly
interesting in the light of previous histochemical and electron
microscopic studies in which acid mucopolysaccharides have been
demonstrated at the surface of a variety of metacestodes (22,23).
In electron microscopic studies we have shown acid-colloidal iron
staining at the surface of larvae of T. taeniaefbrmis (24). This
may be attributable to the occurrence of a surface polyanion in
this parasite, and it is tempting to speculate that the complement-
interacting factors contribute to this layer.

Many other tissue-dwelling parasitic organisms have been
shown to have high densities of negative charges associated with
their outer Surface coats, and these are generally believed to
be polysaccharide in nature. Examples of this can be found among
the helminths [schistosomes (22)], protozoa [Trypanosoma (25,26),
Leishmania (27,28) and amoebae, (29)] and certain strains of bacteria
[Escherichia coli and Salmonella typhi (7)]. In addition, the
surface of trophoblast cells of mammalian embryo and fetal origin

carries a negatively charged component with an affinity for

128
acid-colloidal iron which is believed to be a polysaccharide or
glycosaminoglycan (30,31). While most conclusions on the character-
istics of cell surface constituents of parasites have been drawn
from histochemical studies and lectin binding experiments, in the
case of endotoxin of E. coli and Salmonella origin a molecular
definition of the polyanionic K and Vi antigens has existed for
some time (7). Increasing evidence that polyanions can affect host
amplification systems such as the classical and alternate pathways
of complement fixation and the coagulation cascade (3,4,5,6,32)
suggests that their role on the surface of infectious organisms may
be important in host-parasite relationships.

There is ample evidence that the presence or absence of
complement-fixing factors on the surface of gram-negative bacteria
is the crucial determinant of resistance or susceptibility of the
organism to serum. Reynolds and Pruul have shown that surface
lipopolysaccharides of serum resistant S. typhi buffer complement
consumption in such a way that the cell remains intact (8,33,34).

In a variety of other cell systems important functions have been
ascribed to surface acidic polysaccharides, including adsorption

of other molecules and ions which might then be internalized (35,36),
and the inhibition of intestinal enzymes so that membranes might

be protected against digestion (35,37). It has also been postulated
that the surface glycosaminoglycans of trophoblast cells are involved
in the immunological inertness of the mammalian embryo (30,31,38).
Support for this proposal can be drawn from recent work in which

a correlation has been observed between surface polysulfated

glycosaminoglycan levels on transformed cell lines and their

129
susceptibility to antibody—mediated complement-dependent lysis.
Highest amounts of surface heparan sulfates were present during the
pre-mitotic phase of the cell cycle (39,21) at which time the cells
resisted immunologic lysis, even though antibody binding and complement
fixation continued to occur (40,41,42). Direct evidence of inhibition
of complement-dependent lysis by surface acid mucopolysaccharides
has recently been developed by Lippman (43).

In the host-parasite system which we have studied, an antibody-
mediated immunity develops which is dependent on complement for
effective protection against early larval stages (44) yet larvae
rapidly become insusceptible to this immune mechanism (45). A
protective function for the complement-reactive material in the
larval surface coat can be postulated, analogous to that proposed
by Reynolds, Rother, and Rother (34) for surface lipopolysaccharide
in serumrresistant strains of Salmonella. It may be that the
lethal effect on early stage larvae is due to the ability of the com-
plement-fixing antibodies to concentrate critical amounts of complement
at vital membrane foci. With further development the larvae become
resistant to complement-fixing antibodies, possibly due to local
depletion of complement or inhibition of antibody-binding. Both
of these activities have been described for polyanionic
polysaccharides (7).

The recently reported susceptibility of cestodes to a 1ytic
effect produced by normal serum complement in vitro (46,47) could
be due to overwhelming local complement concentrations with fixation
to surface factors which, under in vivo conditions, are either

released or placed in a position to consume complement away from

130

the membrane. Further sequential studies on the quantitative
aspects of production and release of complement-fixing factors
during development of T. taeniaefbrmis may provide a means to
examine this phenomonen experimentally, and to clarify the role
of surface polyanions in the successful evasion of rejection by

this parasite.

ACKNOWLEDGEMENTS
This work was supported by USPHS grant AID 10842. This is
journal article No. from the Michigan Agriculture Experiment
Station. We are indebted to Miss Sharon Garland for technical

assistance in many aspects of this study.

LITERATURE CITED

1. Lichtenstein, L. M., H. Gewurz, N. F. Adkinson Jr., H. S. Shin,
and S. E. Mergenhagen. 1969. Interactions of the complement
system with endotoxic lipopolysaccharide: the generation of an
anaphylatoxin. Immunology 16:327-336.

2. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1968. Interactions
of the complement system with endotoxic lipopolysaccharide:
consumption of each of the six terminal complement components.

J. Exp. Med. 128:1049-1057.

3. Loos, M., E. Raepple, U. Hadding, and D. Bitter-Suerman. 1974.
Interaction of polyanions with the first and the third component
of complement. Fed. Proc. (Abst.) 33:775.

4. Fiedel, B. A., R. Rent, R. Myhrman, and H. Gewurz. 1976.
Complement activation by interaction of polyanions and polycations.
II. Precipitation and role of IgG, Cl , and Cl-INH during
heparin-protamine-induced consumption of complement. Immunology
30:161-169.

5. Rent, R., N. Ertel, R. Eisenstein, and H. Gewurz. 1975.
Complement activation by interaction of polyanions and polycations.
I. Heparin-protamine induced consumption of complement.
J. Immunol. 114:120-124.

6. Rent, R., R. Myhrman, B. A. Fiedel, and H. Gewurz. 1976.
Potentiation of Cl-esterase inhibitor activity by heparin.
Clin. Exp. Immunol. 23:264-271.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

131

Glynn, A. A. 1972. Bacterial factors inhibiting host defense
mechanisms. Symp. Soc. Gen. Microbiol. 22:75-112.

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