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This is to certify that the

dissertation entitled

The in vitro Effects of Soluble Yeast Mannan on
Wouse Peritoneal Macrophage Funct1on.

presented by

Michael Peter Kolotila

has been accepted towards fulfillment
of the requirements for

Ph.D degreein Department of Botany and
Plant Pathology

 

0-,"

 

Major pro'fessir 0

Alvin L. Roge 5

Date W85—

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

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THE I VITRO EFFECTS OF SOLUBLE NANNAN ON

 

HOUSE PERITONEAL NAOROPHAOE FUNCTION.

aY

Michael Peter Kolotila

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Departnent of Botany and Plant Pathology

1985

.352? —~ 7503'

 

AN ABSTRACT OF A DISSERTATION

THE L_ VITRO EFFECTS OF SOLUBLE YEAST HANNAN 0N
HOUSE PERITONEAL HACRDPHABE FUNCTION.
by

Michael Peter Kolotila

A new method for assessing the candidacidal activity of macrophages
using the supravital stain neutral red is described. This method is
comparable to well established vital staining techniques, methylene blue
and acridine orange, and to the lack of germ tube formation of Candida
albicans as a method of assessing the candidacidal activity of phago-
cytic cells.

This study characterizes the inhibition by Saccharomyces cerevisiae

 

mannan on the phagocytic activity and phagocytic capacity of Concanava-
lin A elicited peritoneal macrophages towards unopsonized Q. albicans
and Q. albicans opsonized with C3. The interference by mannan on phago-
cytosis of C3 opsonized Q. albicans has not been previously reported.
The phagocytic activity and phagocytic capacity of Con A elicited peri-
toneal macrophages towards Q. albicans opsonized with either normal
human serum or immunoglobulins are unaffected by the presence of eannan.
The inhibitory effect of mannan on phagocytic cells' candidacidal ac-
tivity and superoxide generation are confirmed for macrophages and
expanded to demonstrate that the inhibition of these functions is inde-

pendent of the type of opsonization employed. Lastly, this study

 

Michael Peter Kolotila

demonstrates that phagolysosome fusion of macrophages is unimpaired by
the presence of mannan which supports the previous observations that

mannan does not interfere with neutrophil degranulation.

DEDICATION

To my parents, Mike and Vera Kolotila, who encouraged me to learn and

who instilled in me a love of books and learning.

ACKNOWLEDGEMENTS

I would like to first acknowledge my parents, Mike and Vera
Kolotila, without whose unfailing support I might not have not finished
this dissertation. Next I would like to express my appreciated to the
memebers, both past and present, of the Alex B. Filnow Memorial
Symposium. You have made the good times better and the bad times more
bearable. I would also like to thank Amy Douglass, Jim Hollers, Barbara
Heerdt, Julie Stickle and other friends that I made here at HSU for
their friendship, support and providing me with coffee that helped keep
me going.

I would like to thank Dr. A.L. Rogers for serving as my major
professor and for his critical editing of this manuscript. Also, I
would like to thank Drs. 5.5. Beneke, K. Baker, C.N. Smith and R.J.
Patterson for serving on my committee and for their editing of this
manuscript. I would like to express a special thanks to Dr. C.N. Smith
for his generous use of his knowledge and laboratory during the initial

stages of this dissertation's research. Thank you all for your help.

TABLE OF CONTENTS

Table of Figures
Table of Tables
Introduction
Literature Review
Material and Methods
Results

Discussion

Literature Cited

iv

24

32

69

7B

TABLE OF FIGURES

Figure l. Macrophage with neutral red stained yeast cells
within a phagolysosome. 34

Figure 2. Macrophage with numerous yeast cells within a
large phagolysosome showing both neutral red
stained, nonviable, and unstained, viable, yeast

 

cells. 34
Figure 3. Macrophage that illustrates a different staining

pattern for neutral red stained yeast cells. 34
Figure 4. Lymphocytes that have not taken up neutral red. 34

Figure 5. .Candidacidal activity of Con A elicited macro-
phages from female mice as measured by different
candidacidal assays. 38

Figure 6. The influence of different concentrations of
mannan on the phagocytic capacity of Con A
elicited macrophages from female mice using 4.5
x 10‘ unopsonized Candida albicans. 41

 

Figure 7. The influence of different concentrations of
mannan on the phagocytic activity of Con A
elicited macrophages from female mice using 4.5
x 10‘ unopsonized Candida albicans. 43

 

Figure B. The phagocytic capacity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of
mannan for 5, 10, 15, 20, and 30 minutes prior to
addition of 4.5 x 106 unopsonized C. albicans. 46

Figure 9. Phagocytic activity of Con A elicited macrophages
from female mice treated with 1 mg/ml of mannan
prior to the addition of 5.5 x 10‘ C. albicans. 4B

Figure 10. The phagocytic activity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of
mannan prior to the addition of different concen-
trations of C. albicans opsonized with immuno-
globulins. 51

Figure 11. The phagocytic activity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of

V

Figure

Figure

Figure

Figure

Figure

Figure

13.

I4.

15.

16.

17.

mannan prior to the addition of different concen-
trations of C. albicans opsonized with comple-
ment.

The phagocytic capacity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of
mannan prior to the addition of 5.5 x 10° C.
albicans opsonized by different means.

The phagocytic capacity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of
mannan prior to the addition of different concen-
trations of C. albicans opsonized with immuno-
globulins.

The phagocytic capacity of Con A elicited macro-
phages from female mice treated with 1 mg/ml of
mannan prior to the addition of different concen-
trations of C. albicans opsonized with comple-
ment.

The candidacidal activity of Con A elicited macro-

phages from female mice towards 5.5 x 10‘ C.
albicans opsonized by different means.

The percent Con A elicited macrophages from female

mice reducing NBT after the addition of 5.5 x 106
C. albicans opsonized by different means.

Percent fusion of phagolysosome of Con A elicited

macrophages from female mice after the addition of

4.0 x 10‘ normal human serum opsonized C.
albicans.

vi

53

55

58

60

62

65

67

Table 1.

TABLE OF TABLES

Uptake of neutral red and intracellular gern tube
formation by Candida albicans in phagolysosomes
of peritoneal macrophages.

 

vii

35

INTRODUCTION

Host defense against Candida albicans involves both cell mediated

 

 

and humoral immunity as well as nonspecific immune mechanisms (Rogers
and Balish, 1980). Both neutrophils (Diamond, 1981) and macrophages
(Lehrer and Fleischmann, 1982) are capable of destroying C. albicans as

a part of their nonspecific immune function against microbial invasion

 

 

of the host. Macrophages may be stimulated to greater microbicidal
activity against a wide range of microorganisms (Remington, 1982) which
includes C. albicans.

Factors that aid C. albicans in establishing an infection have been
the subject of renewed interest because of the increasing incidence of
this organism as an opportunistic pathogen. Mannan (one of the main
components of the cell wall of C. albicans (Sullivan gt_al:, 1983)) is
one factor that affects not only cell mediated and humoral immunity but
also nonspecific phagocytic cells, i.e. neutrophils and macrophages.

Mannan was isolated from serum of infected individuals by Fischer et_al,
(1978). They were the first to show that this mannan would inhibit
lymphocyte proliferation. This observation was expanded by other inves-
tigators with regard to the interaction of mannan and lymphocytes.
Wright gt _L. (1983) demonstrated that mannan inhibits in a dose
dependent fashion the myeloperoxidase mediated killing of C. albicans by

neutrophils as well as killing in a cell free system.

Macrophages play an important role in the defense against infection

(Horwitz, 1982; Lehrer and Fleischmann, 1982; Murray, 1982; Remington,
1982) but little is known about the effects of mannan on these cells.
Since mannan antigenemia is a finding in invasive candidiasis (Neiner
and Yount, 1976) and macrophages are long lived phagocytic cells which
are found throughout the body, it is important to determine the effects
of mannan on these cells. One of the objectives of this study was to
determine the effects of mannan on Con A elicited mouse peritoneal
macrophages with respect to phagocytosis of C. albicans using three
receptors, mannosyl/fucosyl glycoprotein, F: and C3, alone and in combi-
nations. As part of this objective, the intracellular destruction of Q.
albicans and phagocytosis were measured by a new candidacidal assay,
neutral red. Another objective was to determine the affects of mannan

on superoxide production and phagolysosome fusion by these macrophages.

 

 

LITERATURE REVIEW

Candidiasis is one the oldest diseases known to mankind. Thrush,
an oral Candida infection, was described in debilitated patients by
Hippocrates in his Epidemics (Odds, 1979; Rippon, 1982). The report by
Hippocrates as well as by other early investigators emphasizes a major
point about candidiasis which is its association with debilitated
patients (Odds, 1979; Rippon, 1982). Since Hippocrates' times, Candida
diseases have been the subject of intense scientific interest. These
investigations have reinforced the observations of the intimate associa-
tion between humans and members of the genus Candida, especially
Candida albicans (Bodey and Fainstein, 1985; Odds, 1979; Rippon, 1982).
Candidiasis may be defined as any infection, either primary or secon-
dary, caused by a member of the genus Candida. The term candidosis is
used by Odds (1979) to refer to infections caused by members of the
genera Candida, Torulopsis and Rhodotorula. The term candidiasis will
be used in this work and will refer to the infections caused by members
of the genus Candida.

The genus Candida may be briefly defined as yeast that reproduce by
blastoconidia and may form true hyphae or pseudohyphae, but do not
produce carotenoid pigments, usually do not reduce urea and do not have
a capsule (Rippon, 1982). The concept of the genus Candida has been
enlarged to encompass the genus Torulopsis (Yarrow and Meyer, 1978).

Also, van Uden and Buckley (1970) felt that the separation of these two

genera on the basis that Candida produces true hyphae or pseudohyphae
and that Torulopsis does not, was artificial. The combination of these
two genera has been rejected by other mycologists (McBinnis gt aL.,
1984).

Rithin the genus Candida, C, albicans is the most pathogenic member
though other species in this genus are also pathogenic (Hurley, 1980;
Odds, 1979; Rippon, 1982 ). Hurley (1980) presents a table consisting
of seventeen species of Candida that are commonly associated with humans
and another table consisting of eight more species that are occasionally
isolated from humans. She also presents a list of nine species of
Candida that show virulence for humans. The seven most virulent species

of Candida in descending order are C. albicans, Q. tropicalis, Q.

 

stellatoidea (which may be a variant of C. albicans), C. (Torulogsis)

glabrata, Q. krusei and Q. parapsilosis (Hurley, 1980). Of these seven

 

species C. albicans and Q. tropicalis account for at least 80% of the

yeast isolated from humans (Hopfer, 1985). Candida albicans comprises

 

90% of the yeast isolated from vaginal specimens (Hurley, 1980). Candida
parapsilosis and Q. glabrata account for between 10 and 15% of the yeast
isolated from humans (Hopfer, 1985).

Candida albicans is part of the normal flora of humans, usually

 

being associated with the alimentary tract and eucocutaneous surfaces
(Odds, 1979; Rippon, 1982). The recovery of C. albicans from the human
body increases at least two fold in a patient population as compared to
healthy individuals with the notable exception of the skin which remains

relatively the same regardless of the health of the individual (Odds,

1979). Passage through an infected birth canal may lead to oral can-
didiasis of the neonate. Infection rates have been recorded to be as
high as 18%, but the average appears to be about 4% (Rippon, 1982).

It is because of this intimate relationship that C. albicans has
with humans that Candida antigens are used to assess cellular immunity,
either by lymphocyte transformation (Oppenheim and Schecter, 1976) or by
delayed type hypersensitivity skin tests (Spitler, 1976). Humoral immu-
nity is also involved in this relationship. Antibodies to C. albicans
are found in most individuals without a known prior history of Candida
infection (Bough gg_gL., 1984; Lehnmann and Reiss, 1980; Neiner and
Yount, 1976). Unfortunately, they have not been useful in the diagnosis
of Candida infection because the titers from patients with Candida
infections are often within the same range as those individuals without
a Candida infection (Bough gg gL., 1984; Lehnmann and Reiss, 1980).
Even in vaginal secretions antibodies of the 198 and lgO classes to C.
albicans may be found (Bough gg gL., 1984). It is believed that the
persistence of C. albicans in the gastrointestinal tract generates the
antibody response found in most individuals (Lehnmann and Reiss, 1980).
This is interesting because other microorganisms that also reside in the
gastrointestinal tract in higher numbers than C. albicans do not invoke
similar immune responses (Berg and Savage, 1972; Foo and Lee, 1972).

Since C. albicans is usually part of the normal flora of humans it
has the opportunity to exploit any crack in the homeostatic mechanisms
of its host. Rippon (1982) divided the causes that may predispose an

individual to candidiasis into five major categories: i) extremes in

 

 

 

   

age, ii) physiological change, iii) prolonged administration of anti-
biotics to which immunosuppressive agents may be added, iv) general
debilitation and v) iatrogenic, mechanical and traumatic barrier breaks.
Smith (1985) and Mitchell (1980) have each compiled a list of specific
alterations of the host response that predisposes the host to
candidiasis. The spectrum of disease caused by members of the genus
Candida is quite varied, ranging from superficial colonization, muco-
cutaneous involvement, systemic infection, which may be life threat-
ening, and allergies. Candidiasis leaves no organ system untouched,
being reported to attack almost every part of the body (Bodey and
Fainstein, 1985; Edwards gt_gl,, 1978; Hurley, 1980; Odds, 1979; Rippon,
1982). -

One of the most interesting aspects of any microbial infection is
the mechanism(s) that the microorganism employs to establish an
infection. Since C. albicans is usually part of the normal flora of
its host, an infection by this yeast is termed opportunistic. The
mechanisms that the host uses to keep this microorganism under control
and the mechanisms that the fungus uses to circumvent that control are
an interesting area of study.

Candida albicans appears to employ several mechanisms to establish

 

an infection. The most obvious one and one of the most controversial is
the ability of the fungus to alter its growth form from an yeast like
organism to a filamentous organism. There is still debate as to which
form is the most pathogenic. The majority of investigators have found
that the yeast form is most pathogenic (Evans, 1980; Evans and Mardon,

1977; Mardon gt_gl,, 1975; Rogers and Balish, 1978; Simonetti and

 

 

 

Strippoli, 1973; Vaughn and Heinberg, 1978) though some other investiga-
tors have found the mycelial form to be more pathogenic (Gresham and
Hhittle, 1961; Iannini gt_gl,, 1975; Salterelli gt_gl,, 1975; Young,
1958). Louria gg_gL, (1963) in their study on the pathogenesis of C.
albicans could not make a correlation that either the yeast or mycelial
form of the organism was more invasive. In tissue both forms of the
organism are found (Odds, 1979; Rippon, 1982). In studies concerning
oral candidiasis the necessity of filamentation of C. albicans to pene-
trate the epithelial cells and establish an infection has been demon-
strated (Hewlett and Squier, 1980; Harris and Costerton, 1981; Martin gg
gL., 1984; Montes and Hilborn, 1968). Whittle and Gresham (1960) using
a rabbit model of renal candidiasis found that the production of pseudo-
hyphae was necessary for a fatal infection with C. albicans. If C.

tropicalis, Q. parapilosis or Q. guilliermondi were used, they did not

 

 

form pseudohyphae in the rabbit kidney and thus did not die. However,
if they injected Q. trogicalis into either rats or mice they did die and
pseudohyphae were present. Also, in ig_ggggg_studies Hurley and Stanley
(1969) showed that filamentation of different Candida species caused a
cytopathic effect in cultured cells. It is also this ability to become
filamentous that some investigators believe allows C. albicans to escape
destruction in I'professional" phagocytes (Aral gth13, 1977; Louria and
Brayton, 1964; Louria gg_gg,, 1963; Ozato and Uesaka, 1974; Stanley and
Hurley, 1969).

Once a "professional“ phagocyte comes into contact with C. albicans
as either a yeast form or mycelial form organise, a complex set of

events is set into motion with the result being the destruction of the

fungus. The main mechanism of destruction of Q. albicans by "profes-
sional“ phagocytes is through oxidative metabolism. In patients with a
myeloperoxidase deficiency oxidative metabolism is lacking and their
neutrophils and monocytes do not kill C. albicans while neutrophils and
monocytes from normal donors do kill C. albicans (Lehrer and Cline,
1969). Sasada and Johnson (1980) demonstrated that macrophages killed

C. pargpsilosis more effectively than they did C. albicans. They also

 

found that C. parapsilosis elicited a greater oxidative metabolic

 

response from macrophages than did C. albicans and that compounds which
interfere with oxidative metabolism decreased the killing of both orga-
Inisms. This indicated that the generation of toxic oxygen metabolites
is a major mechanism of intracellular killing of C. albicans (Sasada and
Johnson, 1980). These studies were done with yeast form organisms.
Diamond and Krzesicki (1978) examined the mechanism by which neutrophils
killed the pseudohyphae of C. albicans and found that compounds which
interfere with oxidative .metabolism decreased the killing of the
organism. They found that oxygen metabolites, notably superoxide,
singlet oxygen and hydrogen peroxide acting with myeloperoxidase and
halide, were involved in the destruction of pseudohyphae, but not the
hydroxyl radical. In a latter study, Diamond _g__g. (1980a) further
defined the role of these oxidative metabolites in the destruction of
hyphae and pseudohyphae of C. albicans by neutrophils. They found that
neutrophils from patients with chronic granulamatous disease and myelo-
peroxidase deficiency, both diseases show impaired oxidative metabolism,

and neutrophils treated with inhibitors of oxidative metabolism ex-

hibited a lowered candidacidal activity as compared to the controls.

The important oxidative metabolites, with respect to destruction of
hyphae and pseudohyphae of C. albicans, were hydrogen peroxide, singlet
oxygen and the hydroxyl radical. Superoxide did not play a direct role
in killing but was important in generating hydrogen peroxide (Diamond gg
gL., 1980a). These findings are in conflict with the earlier study with
regard to the hydroxyl radical. This difference was attributed to the
different hydroxyl radical scavengers used in the two studies. In the
second study a much more effective hydroxyl radical scavenger, DMSO, was
used instead of the mannitol and sodium benozate used in the first study
(Diamond _g__g,, 1980a).

If the formation of the hyphal form via germ tubes is a mechanism
by which C. albicans may escape from a phagocytic cell then it would be
expected that the hyphal form of this organism would be more resistant
to killing by phagocytic cells. A study by Culter and Poor (1981) using
ig- gggg_chambers found that the hyphal forms of C. albicans were more
resistant to destruction by neutrophils and macrophages than were the
yeast form cells. The chambers, depending on the membrane pore size,
would allow free diffusion of soluble host substances but would exclude
host phagocytic cells or would allow both soluble host substances and
host phagocytic cells to enter the chamber which housed the C. albicans.
In an gg_!gggg_study, Schuit (1979) found that human blood monocytes
were more effective than human blood neutrophils at killing the hyphal
form of C. albicans. Schuit (1979) also found that the yeast form was
killed to a greater degree than the mycelial form of the organism
regardless of the phagocytic cell type. The differences seen between

the monocytes' and neutrophils' ability to kill the hyphal form of Q.

10

albicans was not due to diffences in phagocytosis between the two phago-
cytic cell types because both forms of the fungus were phagocytized to
an equal degree by both types of phagocytic cells.

However, in ig_ giggg_studies with phagocytic cells from several
body sites, Baccarini _g_gl, (1985) found that the hyphal form of C.
albicans was killed equally as well if not better than the yeast form.
Cockayne and Odds (1984), using an ig_ggtgg_technique, found that the
hyphal form of Q. albicans was more effectively killed than the yeast
form by neutrophils. Both studies used a radiometric assay to detect
the killing of the fungus so quantitation of phagocytosis by the phago-
cytic cells with respect to the two growth forms of C. albicans was not
measured. This is perhaps a moot point because Diamond _g gL.
(1978) demonstrated that neutrophils could damage pseudohyphae that were
too large for them to completely engulf. The neutrophils spread over
the surface of the pseudohyphae and lysosomes were degranulated with

subsequent damage to the pseudohyphae.

Candida albicans possesses a repertoire of enzymes that may aid it

 

in establishing an infection. The most widely studied of these enzymes
are the phospholipases (Banno gg_gl,, 1985; Price and Dawson, 1975;
Sammaranayake gg_gg., 1984) which are believed to aid in the penetra-
tion of the hyphae of C. albicans into epithelial cells. Penetration of
hyphae into epithelial cells have been described by Hewlett and Squier
(1980), Harris and Costerton (1981), and Montes and Hilborn (1968).
Both Marrie and Costerton (1981) and Montes and Hilborn (1968) have
observed epithelial cell damage with the invasion of hyphae from C.

albicans. Howlett and Squier (1980) found little damage to the

11

epithelial cells and what damage was observed was limited to the site of
invasion. The localization of the damage to epithelial cells corre-
sponds to the observations of Pugh and Cawson (1977) who histochemical-
1y located phospholipase in the developing hyphae that had penetrated
the epithelial cells. Samaranayake _g_gl, (1984) found that only C.
albicans produced phospholipase activity while Q. trgpicalis, C.
glabrata and Q. parapsilosis did not. This would be in accord with C.
albicans being more pathogenic than the other three Candida species.
Banno _g_gl, (1985) demonstrated that the mycelial form of C. albicans
produced roughly twice as much phospholipase activity as did the yeast

form. This lends credence to the concept that the mycelial form of C.

albicans is necessary to establish an infection. Candid albicans has

 

at least one proteinase for which there is strong evidence that it
is a virulence factor (Macdonald and Odds, 1983; 1980), out of the
several proteinases that this yeast possesses (Ruchel, 1984). Macdonald
and Odds (1980) detected antibodies to a proteinase in serum and were
able to detect this same proteinase in infected tissue with indirect
immunofluorescence. In a latter study, Macdonald and Odds (1983) de-
veloped a proteinase deficient strain of C. albicans. Hhen this pro-
teinase deficient strain was grown on a medium that induced proteinase
activity it was phagocytized and killed by neutrophils more readily than
its parent strain . However, when both strains were grown on a medium
that did not induce proteinase activity both strains were phagocytized
and killed to the same extent. This is supporting evidence to the

observation made by Mardon t gL. (1975) that the medium used to grow C.

albicans on influenced its virulence. Hhen Macdonald and Odds (1983)

12

injected both strains into mice the proteinase deficient strain was less
pathogenic than its parent strain. Ruchel (1984) cited unpublished
results of an experiment where mice were immunized with a proteinase.
The mice were protected from the proteinase secreting strain of C.
albicans. Ruchel (1984), in reviewing the substrates of these pro-
teinases, found that they attacked a large number of important proteins
of the body. Perhaps one of the most important substrates of this
proteinase in relation to pathogenicity is that it attacks immunoglobu-
lins, particularly all classes of IgA (Ruchel gt_gl,, 1982). Ruchel
(1984) tested seven Candida species for protolytic activity and found

that only C. albicans and Q. tropicalis showed strong protolytic activi-

 

ty while only C. parapsilosis exhibited weak activity and all the other

 

Candida species showed no activity.

Other enzymes have been implicated in the pathogenesis of C.
albicans but little work has been done with these enzymes. Elinov
(1984) has suggested that carbohydrases may play a role in pathogenesis
by removing sugars from large molecules. Hattori gt_gl, (1984) reported
on a keratolytic proteinase activity in C. albicans and postulated that

it may be important in the pathogenesis of the organism i vivo.

 

Pathogenic microorganisms are known for their ability to evade or
suppress in some manner the immune response of the host (Densen and
Mandell, 1980; Botschlich, 1983; Horwitz, 1982; Mackowiak, 1984; Schwab,
1975; Sptiznagel, 1983). These authors cited research illustrating
that the cell wall or cell wall components are involved in the immuno-
suppressive activity of the microorganisms. Since the cell wall is the

first part of the invading organism to come into contact with the immune

13

system of the host, the cell wall or components of the cell wall may
decide the outcome of that incounter. The cell wall of C. albicans or
cell wall components may influence the immune response of the host which
may be an experimental animal.

San-Blas (1982) reviewed the literature concerning the cell walls
of pathogenic fungi in humans. He came to two conclusion: 1) that much
more work is necessary to establish a correlation between fungal cell
wall structure or composition and pathogenicity and 2) that more work is
necessary to determine why two fungi with similar cell wall structure
and composition differ greatly in their respecitive pathogenicities. An

example of_ the second conclusion is C. albicans and Saccharomyces

 

cerevisiae. The latter rarely causes infections in humans (Eng gt_gL.,
1984; Rippon, 1982). San-Blas (1982) was able to classify fungi patho-
genic in humans based on the cell wall classificaiton scheme of
Bartnicki-Oarcia (1968). These fungi fell into two catagories according
to cell wall composition, those with cell wall composed of chitin-glucan
and mannan-glucan (San-Blas, 1982). Respectively, these two groups are

groups 5 and 6 according to Bartnicki-Garcia (1968). Candida albicans

 

is included in group 6, mannan-glucan, along with the other members of
the Cryptococcaceae and the Saccharomycetaceae (San-Blas, 1982).

Bishop g5_ g1, (1960) determined that the main components of the
cell wall of C. albicans are mannan, glucan and chitin. Elorza _t_gl:
(1983) in investigating the regeneration of protoplasts of C. albicans.
determined the amounts of mannan, glucan and chitin in both growth forms

of the organism. Regardless of growth the amount of glucan remained

relatively constant being 71% for the yeast form and 74% for the

14

mycelial form while mannan dropped from 19% in the yeast form to 4% in
the mycelial form. This decrease in mannan was counteracted by an
increase in the chitin content from 9% in the yeast form to 21% in the
mycelial form. A slightly higher mannan content was found in the yeast
form (about 222) by Sullivan _g gg. (1983) and Cassone _g gL. (1979) as
well as a slightly higher glucan content for the yeast form (about 79%)
The increase in the amount of chitin from the yeast form to the mycelial
form is in agreement with the observations of Chattaway gg_ g1. (1968)
who postulated that this increase in chitin content may result in

greater resistance of the mycelial form to the defense mechanisms of the

host allowing for the persistence of the mycelial form 1 vivo.

 

The localization of mannan has been examined by investigators using
a variety of techniques such as lectins (Con A), antibodies to mannan,
biochemical techniques, histochemical techniques and combinations of any
of the above. Montes and Hilborn (1968) in examining the invasion of
oral epithelial cells by C. albicans noted a floccular material that was
present on the extracellular yeast but not on the yeast cells invading
the epithelial cells. They were not sure if this floccular material was
of host or yeast origin but it was being detached during the invasion of
the epithelial cells. They implied that the floccular material might be
mannan based on the histochemical work of Mundkur (1960) on S.
cerevisiae. Histochemically the localization of mannan in the outer
cell wall of Q. albicans has been confirmed by Djaczenko and Cassone
(1971) using TAPO, and Poulain _t_gl, (1978) using the polysaccharide

staining technique of Thiery (1969). Evron and Drewe (1984) utilizing

extraction and histochemical procedures also placed mannan in the outer

15

cell wall. They analyzed the extraction fluid for cell wall components
and found that the extraction fluid for the outer cell wall layer con-
tained mannan and glucan. This outer layer of mannan is apparently
easily shed based on the observations of Montes and Hilborn (1968) and
Poulain _gigg. (1978). They observed that old cultures of C. albicans
had lost their outer cell wall layers which they said contained mannan.
Due to this loss of outer cell wall layers Poulain gt_ g1, (1978)
postulated that this could account for the mannan antigenemia described
by Heiner and Yount (1976) in patients with invasive candidiasis. In
culture filtrates, Diedrich gt_gl, (1984) and Diamond gt.g1: (1980b)
isolated a glycoprotein that contained mannose which both groups thought
to be mannan. Elorza gg gL. (1983) found that during the regeneration
of protoplasts of C. albicans radioactively labelled mannan was not
associated with the protoplast indicating that mannan was not a struc-
tural polysaccharide of the cell wall as are glucan and chitin but that
mannan is a matrix polysaccharide. This study is in agreement with
Cassone _t__l, (1978) who concluded that mannan is located throughout
the cell wall both over it and interwoven into the glucan-chitin
framework.

Mannan is also located next to the plasma membrane as demonstrated
by Poulain gt_ al. (1978). A trilaminar staining pattern using

antibodies to mannan was described by Meister gg_gg, (1977) and Venezia
and Lachapelle (1973). In both cases the staining was absent when the
antibodies were treated with mannan. Tronchin gg_gl: (1981) arrived at

the same conclusion using Con A and a silver staining technique for

polysaccharides.

16

All three of the above mentioned cell wall components have been
shown to affect the immune system. Blucan is a stimulatory agent of the
reticuloendothelial system (DiLuzio, 1975) and this stimulation of
macrophages is seen under ig_vgggg_conditions (Burgaleta gg_gl,, 1978).
Blucan has also been reported to protect mice from experimental Candida
infections (Williams gg_gl,, 1978). Suzuki _t_gl, (1984) demonstrated
that immunizing mice with chitin increases their longevity when chal-
lenged with C. albicans. They also showed that chitin is a stimulator

of oxidative metabolism in neutrophils and macrophages. The observa-

tions of Suzuki t al. (1984) would tend to support the proposal of

Chattaway gg al. (1968) that the chitin in the mycelial form plays a
role in the infection process of C. albicans.

Mannan is the cell wall component most implicated in the
suppression of the immune response. Mannan antigenemia has been
reported in cases of invasive candidiasis by Heiner and Yount (1976).
In a .latter study, Heiner and Coats-Stephen (1979) confirmed, in
patients and an animal model of candidiasis, the existence of mannan
antigenemia in disseminated candidiasis. Segal gt_gl: (1979) in an
attempt to devise a reliable means of antemortum detection of invasive
candidiasis found mannan concentrations ranging from 25 to 540 ng per ml
of serum in autopsy proven cases of candidiasis. Hright gt_gl, (1981)
isolated from a patient with chronic mucocutaneous candidiasis a re-
markable 2.7 mg of mannan per ml of serum.

Mannan appears to be the predominant polysaccharide cell wall

antigen (Summers gg.gg., 1964). Antibodies to mannan can be found in

serum of patients with candidiasis (Chew and Theus, 1967; Lehnmann and

17

Reiss, 1980; Neiner and Coats-Stephens, 1979, Neiner and Yount, 1976 ),
in experimentally infected animals (Meister gt al. 1977; Heiner and
Coats-Stephens, 1979) and in normal uninfected individuals (Lehnmann
and Reiss, 1980; Heiner and Yount, 1976). Circulating antigen-antibody
complexes have been detected in the serum of patients with candidiasis
(Burges gg_gl,, 1983) and immune complexes can be generated by adding
mannan to human serum (Reiss gt_gl,, 1981). Immune complexes have
been implicated in the lethality seen with Candida infections (Kasckin,
1974). He reported that immune mice when injected with Candida species
developed vascular emboli due to immune complex deposition. Patients
with high anticandidal antibody titers and normal complement levels are
found to have dissemianted Candida infections (LaForce gg_ gl,, 1975).
They found that sera from these patients when used to opsonize C.
albicans decreased intracellular killing by the neutrophils regardless
of whether the neutrophils were from the patient or the uninfected indi-
viduals. Ultrastructurally, they found a heavy deposition around the
opsonized yeast cell which seemed to protect it from being killed inside
the neutrophil.

Fischer gt :1. (1978) found that mannan inhibited the ig_ giggg_
Candida antigen induced T cell proliferation. Other investigators have
found that Candida infection inhibits the immune response of T cell
dependent antigens (Valdez gt_gl,, 1984; Vardinon and Segal, 1979).
Later, Segal gg_gl, (1980) demonstrated tht culture filtrates suppressed
the immune response to T cell dependent antigens but not to the extent
of whole yeast cells. The substance which was not defined by Segal gg

l. (1980) may very well be mannan based on the observations of Diedrich

18

gt_al.

(1984) and Diamond gt_gg, (1980b) that mannan is released into
the culture medium by the fungus. Piccolella gg_gl, (1981a) found that
suppressor cell were generated after exposure to a polysaccharide from
C. albicans. They suggested that the suppression occured by two
possible mechanisms: a direct suppression and an interaction with
soluble helper factors. Human peripheral blood lymphocytes were found
to become unresponsive to a second exposure of this polysaccharide but
the unresponsiveness could be overcomed by the addition of fresh mito-
mycin C treated lymphocytes (Piccolella gg_gL., 1981b). Nelson gg_gg.
(1984) observed that mannan inhibits lymphocyte proliferation to a
.variety of antigens including the Candida antigen and that the inhibi-
tion was due to the copper associated with the mannan. If the copper
was removed, then the inhibition of lymphocyte proliferation to all
antigens except for the Candida antigen was abolished. The copper was
used to isolate the mannan. Nelson _g _L. (1984) proposed a different
mechanism for the inhibition of lymphocyte proliferation based on the
observation that mannan stimulates hydrogen peroxide generation by
neutrophils and monocytes. They based this mechanism on the observation
that hydrogen peroxide has been demonstrated to suppress lymphocyte
proliferation ig_ygtgg_(Metzger gg_gl,, 1980).

The invading microorganism must be able to mediate adherence to
the host surfaces. However, the same mechanism that allows adherence to
the host surface may be used by the host to phagocytize the invading
organism (Ofek and Silverblatt, 1982). Mannose mediated adherence of C.
albicans to human buccal cells has been suggested by Sandin gg al.

(1982). Both macrophages and neutrophils possess receptors that allow

19

them to phagocytize unopsonized Q. albicans which is inhibited by mannan
or mannose (Sung gt_gL., 1983; Narr, 1980; Diamond gt_ gt,, 1980b).

Mannan has been shown to inhibit the phagocytosis of zymosan by
macrophages (Sung gt gt., 1983). They also demonstrated that mannan
isolated from different species of yeast differed in their ability to
inhibit the phagocytosis of zymosan. Oda _t gt. (1983) found that a
galactomannan and a rhamnomannan from Sporothrix schenckii inhibited the
phagocytosis of the yeast form of this organism. Diamond _t gt. (1980b)
isolated a substance from the culture medium of Q. albicans which they
identified as mannan which inhibited the attachment of neutrophils to
the pseudohyphae and hyphae of Q. albicans. Prior to this study,
Diamond and Krzesicki (1978) demonstrated that mannan isolated from
serotypes A and B of Q. albicans were able to inhibit the attachment of
neutrophils to the pseudohyphae of Q. albicans. These authors thought
that the dose of mannan necessary to inhibit attachment of Q. albicans

to phagocytic cells tg_vitro was too high (1 mg) for mannan to be an

inhibitor of attachment 1 vivo. Nright t l. (1981) found no inhibi-

 

tion of phagocytosis of zymosan particles opsonized with normal human
serum by neutrophils. Sung gt_gl: (1983) examined the effect of mannan
on the phagocytosis of 198 and C3b opsonized erythrocytes by
macrophages. In both cases no inhibition of phagocytosis was observed
(Sung gt_gl,, 1983).

Hilger and Danley (1980) demonstrated that whole yeast cells
affected the ability of neutrophils to generate hydrogen peroxide. This
effect was dependent on whether or not the yeast cells were viable.

Increasing concentrations of opsonized or unopsonized killed Q. albicans

20

caused increasing amounts of hydrogen peroxide to be released. However,
if opsonized or unopsonized viable Q. albicans were used increasing
concentrations of yeast cells demonstrated decreasing amounts of re-
leased hydrogen peroxide. The authors suggested that viable blasto-
spores somehow were able to neutralize the hydrogen peroxide. Viable
blastospores elicited a greater metabolic response from the neutrophils
than did the killed blastospores as measured by the reduction of nitro-
blue tetrazolium and chemiluminescence. In a letter study, Danley and
Hilger (1981) demonstrated that mannan can stimulate hydrogen peroxide
production and iodination by neutrophils, but that mannose the _major
sugar of mannan inhibits these reactions and reduces oxygen consumption,
carbon dioxide generation and superoxide generation ,by neutrophils.
They were uncertain as to why mannan and mannose should have opposing
effects on the neutrophils. In their study, Danley and Hilger (1981)
reported that Q. albicans is unable to neutralize even low amounts of
hydrogen peroxide so that mannan in the cell wall is not a mechanism for
the reduced hydrogen peroxide generation seen in neutrophils. Nelson gt
_t. (1984) confirmed the observations of Danley and Hilger (1981) that
mannan increases the amount of hydrogen peroxide generated when neutro-
phils are exposed to mannan. Superoxide generation by neutrophils
exposed to mannan was measured by Nelson t al. (1984) and Nright gt

al. (1981) and in both studies, mannan reduced the amount of superoxide.

Wright gt gt. (1981) proposed that mannan acts as a substrate to
scavenge superoxide. This proposal was based on the observations that
mannan reduced the amount of superoxide generated in hypoxanthine and

xanthine oxidase system and when neutrophils were exposed to mannan

21

their superoxide generation was reduced but mannan did not inhibit the
oxygen uptake by the neutrophils. Nelson _t_gt: (1984) demonstrated
that mannan has a superoxide dismutase-like activity and that this
activity is dependent of the association of copper with the mannan.
When they removed the copper from the mannan it lost all superoxide
dismutase-like activity. Danley _t gt. (1983) demonstrated that neutro-
phils modulate the respiration of Q. albicans, as measured by carbon

dioxide release, but this inhibition is only 30% of the respiration of

the yeast cells alone. Candida albicans was shown in their study to be

 

able to generate hydrogen peroxide and in the presence of iodine and
lactoperoxidase the yeast was able to iodinate itself. This is a rather
self-destructive facet of Q. albicans since the myeloperoxidase-halide-
hydrogen peroxide system has been shown to be a potent antimicrobial
system by Klebanoff (1968) and Lehrer (1969). In addition to the gen-
eration of hydrogen peroxide, Q. albicans is capable of generating
superoxide which Danley gt_gt, (1983) thought might be a mechanism by
which the fungus may enhance invasiveness. Hhile hydrogen peroxide and
superoxide are toxic to the fungus they are also toxic to mammalian cell
as well. Danley _t_gt, (1983) postulated that since Q. albicans can
tolerate low levels of hydrogen peroxide and inhibit higher levels of
hydrogen peroxide that would be detrimental to the yeast, then this
could be a mechanism by which the yeast may establish an infection.
Lysosomes have been shown to participate in the killing of S.

cerevisiae (D'Arcy Hart, 1981) and may play a role in the killing of C.

 

albicans. Fontenla de Petrino and Sirena (1984) measured the release of

three lysosomal eznymes from rat peritoneal macrophages and found that

22

with time, after a single exposure to killed Q. albicans blastospores,
the amount of these enzymes released into the supernatant increased up
to six hours and decreased thereafter. Since the yeast were killed
prior to addition to the macrophages the decrease in the release of the
lysosomal enzymes was presumably due to the decrease in the amount of
antigen to which the macrophages were exposed (Fontenla de Petrino and
Sirena, 1984). wright gt_ gt, (1981) also measured the release of
lysosomal enzymes, but their system employed mannan and neutrophils. In
contrast to Fontenla de Petrino and Sirena (1984) they found no differ-
ence in the release of acid phosphatase and beta-glucuronidase. Hright
gt gt. (1981) found a decrease in the amount of myeloperoxidase in the
supernatant 'of neutrophils exposed to mannan as compared to the
controls. They determined that the myeloperoxidase was binding to the
mannan and that the mannan was binding to the neutrophils resulting in
myeloperoxidase being unavailable for reaction in the assayed when the

supernatant was used. They bound mannan to Sepharose beads and exposed
these beads to isolated myeloperoxidase in their assay. The myeloper-
oxidase was associated with the Sepharose beads (Hright gt gt., 1981).
Later, Hright _t gt. (1984) demonstrated that mannan will bind to

neutrophils and that the binding sites for myeloperoxidase and neutro-

t gt. (1983) found that the

phils to mannan were different. Nright
binding of myeloperoxidase to the yeast cell surface is necessary for
the killing of the yeast and that mannan inhibited killing in a dose
dependent manner. They postulated that the way the yeast may escape
killing is that the soluble mannan interferes with the myeloperoxidase

mediated killing by binding the myeloperoxidase away from the cell

23

surface because the mannan-myeloperoxidase complex still retained the
myeloperoxidase activity. Wright _t _t. (1983) suggested that the
superoxide scavenging role of mannan did not play a role in the protec-
tion of Q. albicans from killing. Also, they suggested that the serum
levels of mannan were not sufficient to inhibit killing but that at the
site of infection mannan levels might be high enough to inhibit the
killing of Q. albicans. The binding site of mannan to myeloperoxidase
was also investigated by Wright gt_gt, (1984) who found that the
phosphate attached to the outer chain of mannan was necessary for
binding of myeloperoxidase to the mannan. They also reported that

serine and threonine linked oligosaccharides of the mannan molecule are

necessary for the binding of mannan to the neutrophils.

MATERIALS AND METHODS

Abbreviations

 

Buffer, Dulbecco's phosphate buffered saline, pH 7.2; TCM, M199 +
25mM HEPES; FITC, fluorescein isothiocyanate; Con A Concanavalin A;
EDTA, disodiue ethylenediaminetetraacetic acid; HEPES, N-2-hydroxyethyl-
piperazine-N'-2-ethanesulfonic acid; PEC, peritoneal exudate cells; SDA,
Sabouraud's dextrose agar; NBT, nitroblue tetrazolium.
§ggttg gt Materials

Sigma Chemical Co., mannan (M 3640), NBT, Con A, HEPES, EDTA, neu-
tral red, Nright stain; Allied Chemical Co., acridine orange, methylene
blue; BIBCO, M199; Difco, SDA, trypticase soy broth; U.S. Biochemical,
FITC labelled antiserum to Fc portion of 198 and to C3; American Scien-
tific Products, Pro-Texx.

Mice

 

CBA/J littermate breeding pairs were purchased from the Jackson
Laboratory (Bar Habor, ME). The mice were housed at the Medical Mycolo-
gy Research Laboratory at Michigan State University and were maintained
by brother-sister matings. In the neutral red candidacidal assay
studies both male and female mice from 8 to 16 weeks old were used. In
the mannan studies only female mice from 8 to 16 weeks old were used.
The mice were individually housed and fed standard laboratory chow and

acidified water gt_libitum.

25

Peritoneal Macrophage Monolayer Preparation

Resident peritoneal macrophages were obtained by washing out the
peritoneal cavity of mice with 3 ml of cold TCM with gentle massage to
dislodge any loosely adherent cells. The Con A elicited peritoneal
macrophages were obtained by injecting 100 micrograms of Con A suspended
in 0.1 ml of buffer intraperitoneally and harvesting the PEC four days
later. The macrophages were removed by washing out the peritoneal
cavity with 3 ml of cold TCM with gentle massage to dislodge any loosely
adherent cells. Both types of macrophages were withdrawn from the
peritoneal cavity with an 18 gauge needle and syringe. Then 0.3 ml of
the peritoneal fluid was layered onto 9 x 22 mm glass coverslips. The
number of macrophages from the peritoneal cavity of female mice injected
with Con A layered onto the coverslips was about 1 x 10°. The macro-
phages were allowed to adhere for 45 minutes at 37 C after which the
monolayers were washed in warm buffer, placed into Leighton tubes and
used immediately. Total and differential cell counts of the PEC were
performed. Mice whose PEC contained more than 5% neutrophils were
discarded.

Yeast Culture

 

Candida albicans (MSU-1), a fecal isolate, was maintained by

 

monthly transfers on SDA slants. The inoculum was grown on SDA slants
for 48 hrs at 37 C. A loopful of this culture was aseptically trans-
ferred to a 250 ml flask containing 100 ml of trypticase soy broth
supplemented with 4% D-glucose and incubated for 15 hours at 37 C on a
rotary shaker (180 rpm). This brought the culture to stationary phase.

The yeast cells were harvested by centrifugation and washed twice with

26

sterile saline. The viability of the yeast culture was determined by
the methylene blue dye technique using 0.01% methylene blue. Cultures
with less than 95% viability were discarded.

Opsonization Procedures

 

Four opsonization procedures were utilized to selectively opsonize
the yeast cells. 1) Unopsonized yeast cells were used after being
washed in sterile saline and resuspended in buffer. 2) Yeast cells were
opsonized in normal human serum for 30 minutes at 37 C. The normal
human serum used had an agglutination titer to Q. albicans of 1:2.
These yeast cells were opsonized with both immunoglobulins and comple-
ment (Morrison and Culter, 1981). This was demonstrated by fluorescence
with FITC labelled antiserum to the Fc portion of 198 and to C3. 3)
Yeast cells were opsonized with complement by a modification of the
technique of Race _t gt. (1981). The complement pathways of freshly
collectly normal human serum were inactivated with EDTA (10mM final
concentration). To 10 ml of this inactivated serum 5 grams of heat
killed Q. albicans were added to remove anticandidal antibodies. The Q.
albicans was killed by boiling the culture in saline for 30 minutes.
The yeast cells were washed twice with saline and stored frozen until
used. The mixture of the heat killed yeast cells and inactivated serum
were incubated for 30 minutes on a rotating shaker at room temperature.
The yeast cells were removed by filtration and the serum was stored at
-20 C until used. This serum was used within 2 days of preparation.
The serum was reactivated with MgClz (10mM final concentration). Viable

Q. albicans were added to this reactivated serum and incubated for 30

minutes at 37 C. The yeast cells were washed twice in buffer and

27

resuspended in buffer. Prior to use the selectivity of the opsonization
procedure was screened with FITC labelled antiserum to Fc portion of IgS'
and to C3. In this procedure only the yeast cells exposed to the
anti-C3 antiserum fluoresced. 4) The above procedure was used to
opsonized the yeast cells with immunoglobulins. Normal human serum was
inactivated with EDTA (10mM final concentration). The yeast cells were
washed twice in 20mM EDTA and then the inactivated serum added. The
mixture was then incubated for 30 minutes at room temperature on a
rotating shaker. The yeast cells were washed twice and resuspended in
buffer. Prior to use the selectivity of the opsonization procedure was
screened with FITC labelled antiserum to Fc portion of 198 and to C3.
In this procedure only the yeast cells exposed to the anti-Fc antiserum
fluoresced. In all of the opsonization procedures the yeast cells were

resuspended in buffer to a concentration of 5.5 x 107 (as a stock yeast
suspension) and kept on ice until used.

Candidacidal Assay

 

To the monolayers of resident and Con A elicited peritoneal
macrophages from male and female mice in Leighton tubes 0.9 ml of TCM
and 0.1 ml of a stock yeast cell suspension (4.5 x 107/ml normal serum
opsonized) were added, which completely covered the coverslip. The
macrophages and the yeast cells were allowed to incubate together for 1
hour at 37 C.

A fresh solution of neutral red indicator dye in buffer (10mg/ml)
was made up fresh every day (Hammond gtggt,, 1980). Fifteen minutes

before the end of the incubation period 10 microliters of the neutral

red solution was added per Leighton tube. At the end of the incubation

28

period the coverslip was removed, washed in warm buffer and examined
immediately. One hundred phagocytizing macrophages were scored for the
number of red (dead) and colorless (viable) yeasts.

A duplicate coverslip was removed at the same time as the neutral
red coverslip. The duplicate coverslip was washed in warm buffer, air
dried, fixed with absolute methanol, and stained with Wright’s stain and
mounted with Pro-Texx. One hundred phagocytizing macrophages were
counted for the number of yeast cell with and without germ tubes.

The neutral red technique was compared to two standard candidacidal
assays: acridine orange and methylene blue. The acridine orange method
‘was that of Pantazis and Kniker (1979). A modification of the methylene
blue technique of Schmid and Bruno (1974) was employed where methylene
blue (0.1 ml of a 0.1% solution in buffer) was added to the Leighton
tube 15 minutes before the end of the incubation.

The phagocytic capacity and candidacidal activity of the
macrophages were determinedby the following formulae:

phagocytic capacity = no. of yeast cells/100 phagocytizing macro-
phages

candidacidal activity = no. dead yeast cells/100 phagocytizing
macrophages

Mannan Treatment Experiments

 

Mannan was dissolved in 0.9 ml of TCM in the following amounts 0.0,
0.5, 1.0 and 2.0 mg and added to monolayers of Con A elicited peritoneal
macrophages. The macrophages were incubated with the different mannan
concentrations for 15 minutes at 37 C after which time 4.5 x 10‘
unopsonized Q. albicans were added. The macrophages and the yeast cells

were incubated together for 30 minutes at 37 C. The coverslips were

29

washed in warm buffer and 50 macrophages were scored for the number of
yeast cells per macrophage and the percent macrophages containing yeast
cells. The lenght of time for treatment with 1 mg of mannan prior to
the additon of the yeast cells was determined by incubating the macro-
phages with mannan for 5, 10, 15, 20, and 30 minutes at 37 C. The same
number of yeast cells were added to the Leighton tubes. The macrophages
(50) were scored for the number of yeast cells per macrophage. The
viability of the macrophages was determined by eosin Y dye exclusion
(Mishell gt_gt,, 1980). Detachment of the macrophages from the cover-
slips was determined by examining the culture medium on a hemacytometer.

Phagocytic and Candidacidal Studies

 

Monolayers of Con A elicited peritoneal macrophages were incubated
for 15 minutes at 37 C with either TCM or TCM plus 1 mg of mannan.
After this incubation 5.5 x 10‘ yeast cells (regardless of the opsoniza-
tion procedure) were added to each Leighton tube. The macrophages and
the yeast cells were incubated for 30 minutes at 37 C. Fifteen minutes
prior to the end of this incubation 10 microliters of neutral red in
buffer were added to each Leighton tube. The monolayers were washed in
warm buffer and mounted in a drop of buffer, sealed with fingernail
polish and examined immediately. The macrophages were scored on three
parameters modified form Simpson t al. (1979). They are:

phagocytic activity I (no. macrophages with yeast cells/100
macrophage) x 100

phagocytic capacity = no. of yeast cells/100 macrophages

candidacidal activity = no. of dead yeast cells/phagocytic
capacity

30

Mannan and [g and Complement Receptors

 

 

The effects of mannan on these receptors were determined by using
decreasing concentrations of Q. albicans opsonized with either
immunoglobulins or complement. The concentrations of yeast cells were 5
x 10‘, 2.5 x 10‘, and 1.25 x 106 per ml. The monolayers of Con A
elicited peritoneal macrophages were incubated for 15 minutes at 37 C
either with TCM or TCM plus 1 mg of mannan prior to the addition of the
yeast cells. The macrophages and the yeast cells were incubated
together for 30 minutes at 37 C. The coverslips were washed in warm
buffer, air dried, fixed in absolute methanol, and stained with Hright's
stain. The coverslips were mounted with Pro-Texx and 100 macrophages
were scored for their phagocytic activity and phagocytic capacity.

NBT Reduction

 

A stock solution of nitroblue tetrazolium was prepared by a
modification of the technique of Cline (1981). A 0.1% stock solution of
NBT in TCM was prepared, filter sterilized and stored at 4 C until used.
The monolayers of Con A elicited peritoneal macrophages were incubated
for 15 minutes at 37 C either with TCM or TCM with 1 mg of mannan. This
medium was removed from the Leighton tubes and replaced with the
appropiate TCM (with or without mannan), normal human serum opsonized Q.
albicans (5.5 x 10 6) and NBT stock solution to yield a final concen-
tration of NBT of 0.05%. The macrophages and the yeast cells were
incubated for 30 minutes at 37 C. The coverslips were then removed from
the Leighton tubes, washed in warm buffer, air dried, fixed with abso-
lute methanol and counterstained with 0.2% safranin O. The coverslips

were mounted with Pro-Texx and 100 phagocytizing macrophages were scored

31

for the presence or absence of a dark blue precipitate which indicates
the reduction of the NBT (Cline, 1981).

Phagolysosome Fusion

 

The Con A elicited peritoneal macrophages were allowed to adhere
for 1 hour to glass coverslips at 37 C. The monolayers were washed with
warm buffer and then labelled with acridine orange according to the
method of Kielian and Cohn (1980). The monolayers were washed in warm
buffer and simultaneously incubated with either TCM or TCM with 1 mg of
mannan and 5 micrograms of acridine orange for 15 minutes at 37 C. The
macrophages were washed with warm buffer and placed into Leighton tubes
to which TCM or TCM with 1 mg of mannan and 4 x 10‘ of normal human
serum opsonized Q. albicans were added. This concentration of yeast
cells yielded 4-5 yeast cells per macrophage. The macrophages and the
yeast cells were incubated together for 30 minutes at 37 minutes. Then
the coverslips were washed in warm buffer, mounted in a drop of buffer,
sealed with fingernail polish and examined with a Zeiss epifluorescent
microscope. One hundred phagocytizing macrophages were scored for
fluorescent and nonfluorescent yeast cells. Fluorescent yeast cells
indicated phagolysosome fusion.

Statistics

 

The statistical analyses were done using the paired Student's “t“
test and an one way ANOVA (comparison of the different candidacidal
assays). The data were transformed accordingly: percentages by the
arcsin square root of the percentage and distribution by the square
root for the statistical analyses but the data are presented in the

table and figures as nontransformed data.

RESULTS

Figure 1 illustrates that neutral red concentrates in the lysosomes
of macrophages. In the macrophage with an arrow in Figure 1 three
neutral red stained Q. albicans yeast cells are inside a phagolysosome.
Figure 2 illustrates a macrophage with numerous ingested yeast cell some
of which are uniformly stained with neutral red indicating that they are
nonviable while viable yeast are not stained or only have their vacuole
stained with neutral red. Next to this macrophage is another macro-
phage that has not ingested any yeast but has its lysosomes stained with
neutral red. The macrophage with small arrow in Figure 3 illustrates a
variation on the staining pattern of nonviable yeast cells. The yeast
is stained uniformly but a central circular structure is not as intense-
ly stained. The macrophage with large arrow in Figure 3 presents
neutral red stained yeast cells and neutral red stained lysosomes. The
lymphocytes with arrows in Figure 4 have not taken up neutral red to an
appreciable degree and usually do not stain with neutral red. Viable Q.
albicans within macrophages may produce germ tubes as seen in Figure 4
(large arrows).

There was an uniform degree of phagocytosis of Q. albicans
opsonized with normal human serum (Table l.) which permitted the post
phagocytic events to be clearly studied. The phagocytic capacity (num-
ber of yeast cells per macrophage) was not statistically different

between all four groups of macrophages. The phagocytic capacity of

32

 

Figure

Figure

Figure

Figure

33

Macrophage with neutral red stained yeast cells within a
phagolysosome. Insert: a macrophage with neutral red
concentrated in the lysosomes.

Macrophage with numerous yeast cells within a large
phagolysosome showing both neutral red stained, nonviable,
and unstained, viable, yeast cells.

Macrophage that illustrates a different staining pattern for
neutral red stained yeast cells (small arrow). A macro-
phage with both neutral red stained yeast cells and lyso-
somes (large arrow).

Lymphocytes (small arrows) that have not taken up neutral
red. Macrophages (large arrows) that have ingested yeast
cells which have formed germ tubes.

 

 

35

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36

resident macrophages from female mice was 3.44, 3.46 for resident macro-
phages from male mice, 3.64 for Con A elicited macrophages from female
mice and 3.88 for Con A elicited macrophages from male mice. The can-
didacidal activity of the resident macrophages from male and female mice
was the same regardless if measured by the .neutral red
technique (32.8% and 34.3%, respectively) or by lack of germ tube forma-
tion (34.3% and 32.0%, respectively). The candidiacidal activity of
Con A elicited macrophages from male and female mice was the same
whether measured by neutral red (80.5% and 82%, respectively) or by lack
of germ tube formation (78.6% and 80.1%, respectively). When the can;
didacidal activity of resident macrophages from female mice (34.3% as
measured by neutral red) was compared to the candidacidal activity of
Con A elicited macrophages from female mice (82% as measured by neutral
red) a statistically significant difference (P>0.01) is seen. The same
observation was made when the candidacidal activity of resident macro-
phages from male mice (32.8% as measured by neutral red) was compared to
the candidiacidal activity of Con A macrophages from male mice (80.5% as
measured by neutral red), that a statistically significant difference
(P>0.01) is seen.

Neutral red was a comparable candidacidal assay method to other
methods, methylene blue and acridine orange, which require the differen-
tial staining of yeast cells to determine viability. Figure 5 illus-
trates this point. The candidacidal activity of macrophages was 74.2 %
when measured by neutral red and methylene blue and 74.9% when measured
by acridine orange. These results are not statistically different.

Mannan did not affect the viability of Con A elicited macrophages

 

Figure

5.

37

Candidacidal activity of Con A elicited macrophages from
female mice as measured by different candidacidal assays.
NR represents neutral red (10 mg/ml of buffer), AO
represents acridine' orange (l:10,000 in buffer), MB
represents methylene blue (0.1% in buffer).

38

 

 

 

 

 

1.
A0

 

 

 

 

100 ..

. p
0 O O
4 2

8O -
60 -

2.00 «23> ueoo 2.020..

Assay

39

at any concentration or incubation times over 15 minutes. The viability
of the control macrophages and the macrophages exposed to 0.5, 1.0 and
2.0 mg of mannan remained at 98% as determined by eosin Y exclusion.
Incubation of the macrophages with 1 mg of mannan for 20 and 30 minutes
did not alter their viability (100% and 98% viability, respectively).
At no time during the incubation period with different concentrations
of mannan or incubation for different lenghts of time with 1 mg of
mannan, nor with the control macrophages was there any detachment of the
macrophages from the glass cover slips.

The inhibition of the phagocytic capacity (number of yeast
cells/macrophage) of the macrophages towards unopsonized Q. albicans by
mannan was seen to progress in a dose dependent manner (Figure 6). The
lowest concentration of mannan ,0.5 mg, exhibited the least amount of
inhibition of the phagocytic capacity (68% of the control) while the
highest concentration ,2.0 mg, showed the most inhibition at 24% of the
control and 1 mg of mannan showed an intermediate amount of inhibition
at 30% of the control.

The inhibition of the phagocytic activity (percent macrophages with
yeast cells) of the macrophages towards unopsonized Q. albicans by
mannan was also seen to progress in a dose dependent manner (Figure 7).
The least amount of inhibition by mannan on the phagocytic acitivity was
seen with 0.5 mg of mannan which was 71% of the control, and the most
inhibition was exhibited by the highest concentration of mannan at 36%
of the control while 1 mg of mannan showed and intermediate amount of
inhibition at 46% of the control.

Since the time period of treatment with mannan was arbitrarily set

Figure

6.

40

The influence of different concentrations of mannan on the
phagocytic capacity (number of yeast cells per macrophage)
of Con A elicited macrophages from female mice using 4.5 x
10‘ unopsonized Q. albicans. The line is the control value
and the open bars are the different mannan treatment
values. Each data point represents at least two
experiments.

41

 

 

 

 

 

 

 

 

 

 

 

 

 

l.—
t.
L 1 e
‘0. o If;
'- q— o

afieqdomafl Jed snag iseeA ;o ieqwnN

2.0

1.0

0.5

mg of Mormon

Figure

7.

42

The influence of different concentrations of mannan on the
phagocytic activity (percent macrophages containing yeast
cells) of Con A elicited macrophages from female mice using
4.5 x 10‘ unopsonized Q. albicans. The line is the control
value and the open bars are the different mannan treatment
values. Each data point represents at least two
experiments.

43

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44

at 15 minutes it was necessary to determine the effect of length of time
of treatment with 1 mg/ml of mannan on the phagocytic capacity of macro-
phages towards unopsonized Q. albicans (Figure 8). The effect of the
length of time of mannan treatment did not vary greatly from 5 to 20
minutes. The inhibition of the phagocytic capacity of the macrophages
at times of 5 to 20 minutes was about 49% of the control. Only at 30
minutes was there a decrease in the phagocytic capacity larger (30% of
the control) than previously seen. On the basis of these experiments, an
incubation time of 15 minutes at 37 C with 1 mg of mannan prior to the
addition of the yeast cells, regardless of the opsonization procedure,
.was adopted.

The effects of mannan on the phagocytic activity of macrophages
towards Q. albicans opsonized by different means is shown in Figure 9.
The phagocytic activity of macrophages towards Q. albicans opsonized
with normal human serum was not affected by the presence of mannan,
96.9% for the control macrophages and 96.1% for the mannan treated
macrophages. The phagocytic activity of the control macrophages being
96.9% and the mannan treated macrophages phagocytic activity being 96.1%
towards Q. albicans opsonized with immunoglobulins. Mannan affected
the phagocytic activity of these macrophages to a statistically sig-
nificant degree (P>0.01). The mannan treated macrophages had a phago-
cytic activity of 91.7% as compared to the controls which had a phago-
cytic activity of 92.9% towards Q. albicans opsonized with complement.
The phagocytic activity of these macrophages was also decreased by a
statistically significant amount (P>0.05). The greatest difference was

seen when unopsonized Q. albicans were employed to measure the effect of

Figure 8.

45

The phagocytic capacity (number of yeast cells/macrophage) of
Con A elicited macrophages from female mice treated with 1
mg/ml of mannan for 5, 10, 15, 20, and 30 minutes prior to
the addition of 4.5 x 10° unopsonized Q. albicans. The line
is the control value and the open bars are the mannan
treatment values. Each data point represents at least two
experiments.

2.0

 

46

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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9.

47

Phagocytic activity (percent macrophages containing yeast
cells) of Con A elicited macrophages from female mice
treated with 1 mg/ml of mannan prior to the addition of 5.5
x 106 Q. albicans opsonized by various means. The shaded
bars represent control values and the open bars are the
mannan treatment values. Each bar represents at least
seven experiments. One * represents P>0.05 and two *
represents P>0.01. UY = unopsonized yeast cells, NHS 8
normal human serum opsonized yeast cells, C3 = yeast cells
opsonized with complement, lg = yeast cells opsonized with
immunoglobulins.

48

 

 

 

  

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NHS

UY

Yeast Treatment

49

mannan on the phagocytic activity of macrophages. The phagocytic ac-
tivity decreased from the control value of 75.6% to 53.5% for the mannan
treated macrophages. This is a statistically significant difference
(P>0.05).

Since the differences between the means of the phagocytic activity
of the macrophages, whether or not treated with mannan, towards Q.
albicans opsonized with either complement or immunoglobulins was small,
this was examined in closer detail using dilutions of the yeast cells.
Under these conditions the inhibitory effect of mannan on the phagocytic
activity of macrophages towards Q. albicans opsonized with
himmunoglobulins was abolished (Figure 10). The phagocytic activity of
the control macrophages and the mannan treated macrophages at S x 106
was 91% and 88%, respectively, at 2.5 x 106 was 65% and 653,
respectively, and at 1.25 x 10‘ was 43% and 44%, respectively. However,
with Q. albicans opsonized with complement the decrease in the
phagocytic capacity of the macrophages remained (Figure 11). The
phagocytic activity of the macrophages treated with mannan was 82% of
the control at 5 x 10‘, 65% of the control at 2.5 x 10‘, and 57% of the
control at 1.25 x 10‘. The inhibition of the phagocytic activity by
mannan was statistically significant at each concentration (P>0.01).

Hannan treatment had the greatest effect on the phagocytic capacity
of macrophages towards unopsonized Q. albicans (Figure 12). The phago-
cytic capacity was reduced from 3.19 yeast cells per macrophage for the
control macrophages to 1.48 yeast cells per macrophage for the mannan
treated macrophages (P>0.01). hannan treatment had no statistically

significant effect on the phagocytic capacity of macrophages towards Q.

Figure

10.

50

The phagocytic activity (percent macrophages containing
yeast cells) of Con A elicited macrophages from female
mice treated with 1 mg/ml of mannan prior to the addition
of different concentrations of Q. albicans opsonized with
immunoglobulins. The shaded bars represent the control
values and the open bars represent the mannan treatment
values. Each bar represents seven experiments.

51

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

80

0 0
6 4

«:00 ammo> 9.33200 emucaocuaz Eoocea

20
0

[X106]

lg Opsonized C.albicans

Yeast Concentration

Figure

11.

52

The phagocytic activity (percent macrophages containing
yeast cells) of Con A elicited macrophages from female
mice treated with 1 mg/ml of mannan prior to the addition
of different concentrations of Q. albicans opsonized with
complement. The shaded bars represent the control values
and the open bars represent the mannan treatment values.
Two * indicates a P>0.01. Each bar represents eight
experiments.

53

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

80
60
40

2.00 33> 9:53:00 mongoose: 2.805..

O 0
2

Yeast Concentration [x 106]

C3 Opsonized C.albicans

Figure 12.

54

The phagocytic capacity (number of yeast cells/macrophage)
of Con A elicited macrophages from female mice treated with
1 mg/ml of mannan prior to the addition of 5.5 x 10‘ Q.

albicans opsonized by different means. The shaded bars are

the control values and the open bars are the mannan
treatment values. Two * indicate a P>0.01. Each bar
represents at least seven experiments. UY = unopsonized
yeast cells, NHS = normal human serum opsonized yeast
cells, C3 = yeast cells opsonized with complement, 19 =
yeast cells opsonized with immunoglobulins.

 

    
    

   

 

   
    
 

 

 

   

 

         

   
   

     
    

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55

 

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emu—30322 .oo 3.00 53> .o 39:32

Yeast Treatment

56

albicans opsonized with normal human serum, complement and
immunoglobulins (Figure 12). The phagocytic capacity of macrophages,
control and mannan treated, towards Q. albicans opsonized with normal
human serum are 4.45 and 4.23 respectively, for complement opsonized Q.
albicans are 4.76 and 4.85 respectively, and for immunoglobulin opso-
nized Q. albicans are 5.56 and 5.39.

The phagocytic capacity of macrophages towards Q. albicans
opsonized with complement and immunoglobulins at different yeast cell
concentrations was also measured. The presence of mannan had no effect
on the phagocytic capacity of macrophages towards immunoglobulin
opsonized Q. albicans (Figure 13). The phagocytic capacity_of these
macrophages, control macrophages and mannan treated macrophages, was for
3.50 and 3.34, respectively with 5 x 10°, 1.57 and 1.57, respectively
with 2.5 x 10° and 0.80 and 0.79, respectively for 1.25 x 10°. In
contrast to the previous finding that mannan had no effect on the
phagocytic capacity of macrophages towards complement opsonized Q.
albicans (Figure 12) when dilutions of the complement opsonized yeast
cells were employed a statistically significant difference (P>0.01 at
each concentration) was detected (Figure 14). At 5 x 10° yeast cells
the inhibition of the phagocytic capacity of macrophages by mannan was
56% of the control while 2.5 x 10° and 1.25 x 10° the inhibition was 38%
and 43% of the control, respectively.

The candidacidal activity of macrophages exposed to mannan was
decreased, regardless of the opsonization procedure utilized, as
compared to the controls (Figure 15). The greatest inhibition of the

candidacidal activity of the macrophages by mannan was seen when 9.

 

Figure

13.

57

The phagocytic capacity (number of yeast cells/macrophage)
of Con A elicited macrophages from female mice treated
with 1 mole] of mannan prior to the addition of different
concentrations of Q. albicans opsonized with
immunoglobulins. The open bars represent the control
values and the shaded bars represent the mannan treatment
values. Each bar represents seven experiments.

Number of Yeast Cells per Macrophage

58

 

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Yeast Concentration [x106]

lg Opsonized C. albicans

Figure

14.

59

The phagocytic capacity (number of yeast cells/macrophage)
of Con A elicited macrophages from female mice treated
with 1 mg/ml of mannan prior to the addition of different
concentrations of Q. albicans opsonized with complement.
The shaded bars represent the control values and the open
bars represent the mannan treatment values. Two *
indicate a P>0.01. Each bar represents eight
experiments.

60

 

 

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Yeast Concentration [x106]

 

C3 Opsonized C. albicans

 

Figure 15.

61

The candidacidal activity of Con A elicited macrophages from
female mice towards 5.5 x 10° 9. albicans opsonized by
different means. The shaded bars represent the control.
values and open bars represent the mannan treatment values.
Two * indicates a P>0.01. Each bar represents eight
experiments. UY = unopsonized yeast cells, NHS = normal
human serum opsonized yeast cells, CS = yeast cells
opsonized with complement, lg = yeast cells opsonized with
immunoglobulins.

62

 

 

 

 

   

 

 

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Yeast Treatment

63

albicans was opsonized with normal human serum at 75% of the control
(P>0.01). The inhibitory effect of mannan of the candidacidal activity
of macrophages towards Q. albicans opsonized with complement or
immunoglobulins or unopsonized Q. albicans was 80%, 81% and 791 of the
control value, respectively (P>0.01 for each opsonization).

hannan had an inhibitory effect on the superoxide present in macro-
phages which had ingested Q. albicans that were opsonized by different
means (Figure 16). The pattern of inhibition of the reduction of su-
peroxide did not follow the same pattern as seen in the reduction of the
candidacidal activity of the macrophages (Figure 15). The least amount
of reduction of superoxide was seen in macrophages which had ingest Q.
albicans opsonized with normal human serum at 73% of the control
(P>0.01) and the most reduction of superoxide was seen with macrophages
which had ingested Q. albicans opsonized with immunoglobulins at 43% of
the control (P>0.05). Unopsonized and complement opsonized Q. albicans
showed an intermediate reduction of superoxide at 55% of the control
(P>0.05) and 532 of the control (P>0.01), respectively.

The effects of mannan on the phagolysosome fusion of macrophages
was examined using acridine orange and Q. albicans opsonized with normal
human serum (Figure 17). The macrophages were incubated with Q.
albicans so that differences in phagocytosis would not influence the
assay because mannan did not have an effect on the phagocytic capacity
of macrophages. The phagocytic capacity of the control macrophages and
mannan treated macrophages was 3.81 and 3.72 yeast cells per macrophage,
respectively. There was no statistical difference between these two

values. Mannan was determined to have no effect on the phagolysosome

Figure

16.

64

The percent Con A elicited macrophages from female mice
reducing NBT after addition of 5.5 x 10‘ Q. albicans
opsonized by different means. The shaded bars represent
the control values and the open bars represent the mannan
treatment values. One * indicates a P>0.05 and two a
indicates a P>0.01. Each bar represents at least
five experiments. UY = unopsonized yeast cells, NHS =
normal human serum opsonized yeast cells, C3 = yeast cells
opsonized with complement, lg = yeast cells opsonized
with immunoglobulins.

65

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66

Percent fusion of phagolysosomes of Con A elicited
macrophages from female mice after the addition of 4.0 x~
106 normal human serum opsonized Q. albicans. The shaded
bar is the control value and the open bar is the mannan
treatment. Each bar represents five experiments.

67

 

 

 

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68

fusion using the acridine orange technique. The percent fusion of
phagosomes and lysosomes was 85.5% for the control macrophages and 84.1%

for the mannan treated macrophages.

DISSCUSIDN

The use of neutral red as a candidacidal assay is described in this
study. Neutral red was chosen for the candidacidal assay for three
reasons. First, neutral red accumulates in the lysosomes of macro-
phages, as well as in other cell types, such as fibroblasts, neutro-
phils, mast cells, and eosinophils (Bulychev gt_al,, 197B; Cohn and
Wiener, 1963; Hammond gt_al,, 1980; Ogawa, 1961). Secondly, neutral
-red exhibits low toxicity towards macrophages (Hammond gt. gL., 1980).
Lastly, neutral red has been used to determine the viability of yeast
cells (Hills, 1941). The staining pattern is the same as seen by Hills
(1941) where the nonviable yeast cells are uniformly stained red and
only the vacuole of viable yeast cells stained red (Figures 1-4). A
second staining pattern for nonviable yeast cells is described where the
yeast cells are uniformly stained red except for a circular central
structure which is not intensely red (Figure 3).

The use of neutral red to determine the candidacidal activity of
macrophages is comparable to other methods that depend on the differen-
tial staining of yeast cells (Figure 5). The other methods are acridine
orange (Pantazis and Kniker, 1979) and methylene blue (Lehrer and Cline,
1969; Schmid and Brune, 1974). Methylene blue is dependent on the
metabolic state of the yeast cell since metabolically inactive yeast
cells stain a light blue similar to dead yeast cells with no RNA

(Lehrer, 1981). Acridine orange is a rapid and simple method to deter-

69

70

mine the microbicidal activity of macrophages (Pantazis and Kniker,
1979) but it requires an UV microscope while both methylene blue and
neutral red require only a light microscope. Also, acridine orange is a
mutagen as shown by the Ames assay (McCann gt_al,, 1975). The lack of
germ tube production has been used to measure the candidacidal activity
of phagocytic cells and in this study, neutral red was demonstrated to
be comparable to this technique (Table 1).

These studies were designed to examine the influence of soluble Q.

cerevisiae mannan on the macrophage’s ability to ingest and kill selec-

 

tively opsonized Q. albicans yeast cells and on the intermediate events
of phagolysosome fusion and superoxide generation. One of the first
method(s) by which mannan may affect the macrophage is toxicity. It was
found in this study that mannan was not toxic to macrophages in concen-
trations up to 2 mg/ml nor was it toxic at 1 mg/ml when incubated with
macrophages for up to 1 hour. This is in agreement with the observa-
tions by Stahl and Gordon (1982) who reported that 2 mg/ml of mannan
were not toxic to macrophages or their hybrids even after 48 hours of
culture.

There are three receptors on the surface of macrophages that have
been implicated in host defense against Q. albicans. They are the
mannosyl/fucosyl glycoprotein receptor (Harr, 1980), the Fc receptor and
the C3 receptors (Ferrante and Thong, 1979; Morrison and Culter, 1981;
Pereira and Hosking, 1984; Schmid and Brune, 1974). The present study
demonstrates that Con A elicited mouse peritoneal macrophages, like

mouse alveolar macrophages (Narr, 1980), are capable of phagocytizing

71

unopsonized Candida yeast cells. Inhibition of phagocytosis of unopso-
nized Q. albicans, whether measured by phagocytic activity (Figures 7
and 9) or by phagocytic capacity (Figures 6, 8, and 10) by macrophages
is depressed in the presence of mannan. Hannah may accomplish this
depression of phagocytosis in two ways. The first method being the
physical blocking of the receptor so that the unopsonized yeast cell
cannot bind to the receptor. The second method is that mannan causes
the internalization of the mannosyl/fucosyl glycoprotein receptor which
perphaps is the most likely method. Sung gt_alJ (1985) cite unpub-
lished observations that Q. cerevisiae mannan increases the fluid phase
pinocytosis of thioglycollate elicited macrophages which result may
internalation of this receptor.

hannan was found not to interfere with the phagocytosis of normal
human serum opsonized Q. albicans by macrophages (Figures 9 and 12).
This is consistant with the findings of “right _3 _L. (1981) that mannan
not does interfere with the phagocytosis of serum opsonized zymosan. It
is unclear whether or not the opsonzied zymosan used by Wright gt_ aL.
(1981) was opsonized with both immunoglobulins and complement. It is
likely that the zymosan was opsonized with immunoglobulins and comple-
ment based on the observation of Roos _g_aL. (1981) who found immuno-
globulins to zymosan in pooled normal human serum.

Collectively, there appeared to be no decrease in the phagocytosis
of normal human serum opsonized Q. albicans by macrophages in the pres-
ence of mannan. This does not mean that either the Fc or C3 receptors

were not affected or that the one receptor is compensating for the

other. Hhen immunoglobulin or complement opsonized Q. albicans cells

72

were incubated with macrophages that had been treated with mannan a
decrease in the phagocytic activity was seen (Figure 9). The differ-
ence between the control and mannan treated macrophages was small but
statistically significant. Further studies using decreasing concen-
trations of selectively opsonized Q. albicans were undertaken to deter-
mine if this was a real event (Figures 10 and 11). The results of these
experiments with the Fc receptor were in contrast to results obtained by
Sung gt_al, (1983) who found no difference in phagocytosis of 198 coated

erythrocytes by macrophages plated onto 5. cerevisiae mannan coated

 

coverslips as compared to control macrophages. Further studies did not
confirm the earlier results for the differences in the phagocytic ac-
tivity of the control versus the mannan treated macrophages the de-
creasing concentrations of Q. albicans. In neither case (Figures 12
and 13) was a decrease of the phagocytic capcaity of the macrophages
seen in the presence of mannan. In a later study, Sung t al. (1985)

found that Kloeckera brevis mannan was capable of causing a decrease in

 

the phagocytosis of IgG coated erythrocytes by macrophages. They sug-
gested that K. brevi§_ mannan has a much higher affinity for the
mannosyI/fucosyl glycoprotein receptor than does §. cerevisiae mannan
and that the g. brevis mannan caused the internalization of the
mannosyl/fucosyl glycoprotein receptor and the co-internalization of the
Fc receptor. The results of Sung _t at, (1985) suggest that these
experiments be repeated using Q. albicans mannan as it may have a higher

affinity for the mannosyl/fucosyl glycoprotein receptor than does §.

cerevisiae mannan.

 

The decrease in the phagocytic activity (Figure 9) of macrophages

73

phagocytizing C3 opsonized Q. albicans in the presence of mannan was
very small as compared to the control macrophages. Additional studies
using decreasing concentrations of C3 opsonized Q. albicans demonstrated
that mannan can interfere with the phagocytic activity of macrophages to
a very large degree (Figure 11) and that this interference can be ex-
tended to the phagocytic capacity (Figure 14) of macrophages where an
interference was not seen before (Figure 12). These results are in
contradiction to those obtained by Sung _t__l, (1983) who demonstrated
no difference in the phagocytosis of C3 coated erythrocytes by thio-
glycollate elicited peritoneal macrophages plated on mannan coated
coverslips as compared to the control macrophages. The most likely
explanation for this difference in results is that two different C3
receptors were being examined. Macrophages have several different C3
receptors on their surface (Fearon and Wong, 1983). In neither study,
this one or Sung gt_aL. (1983), was the type of C3 split product de-
posited on the particle surface identified. It may be that Sung _t al.
(1983) were examining the CR1 receptor which has its highest affinity
for C3b and the present study examined the CR3 receptor which has its
highest affinity for iC3b. The reason that CR3 receptor is suspected of
being inhibited in this study is based on the results of Ross _t_aL.
(1985) who demonstrated that the CR3 receptor is composed of two
functional sites. One site binds iC3b and the other is a lectin-like
site that binds unopsonized zymosan and §. cerevisiae. The implications
of this complement receptor having two binding sites is intriguing.

Zymosan and yeast cells are capable of activating complement (Smith gt_

£13, 1982). It is possible that both sites on the CR3 receptor function

 

74

in the binding and ingestion of iC3b coated yeast cells so that blockage
of the Iectin-like site would inhibit ingestion. This is suggested by

the results of Ross gt al. (1965) where they demonstrated that mannan

(20 mg/ml) did not interfere with the binding of iC3b coated erythro-
cytes to neutrophils. However, they did test the mannan against the
lectin-like site which is the site that is involve in the ingestion of
particles while the iC3b is important in binding. Mannan may function
to promote the infection by inhibiting the ingestion of the yeast cells
by phagocytic cells even though the yeast cells are bound to the phago-
cytic cells by the iC3b site of the CR3 receptor. The questions of
whether or not this lectin-like component of the CR3 receptor is dif
ferent from the mannosyl/fucosyl glycoprotein receptor or if it is able
to bind and promote the uptake of mannan have yet to be answered. There
remains a great deal of work necessary to clarify the interaction of
mannan, especially from Q. albicans, and the Fc and complement recep-
tors.

Con A elicited peritoneal macrophages are capable of a high degree
of intracellular killing of Q. albicans yeast cells regardless of the
type of opsonization of the yeast cells (Figure 15). This indicates
that these macrophages have only one level of candidacidal activity
which is not influenced by the opsonization of the yeast cells. These
results are in conflict with the results presented by other investiga-
tors (Pereira and Hosking, 1984; Schmid and Bruno, 1974; Yamamura and
Valdimarsson, 1977) who reported that complement was necessary for
maximum killing of Q. albicans. The difference between their results

and these results may be the different phagocytic cell types used,

75

neutrophils versus macrophages, respectively. Mannan reduced the
intracellular killing ability of macrophages to a similar extent
regardless of the opsonization of the yeast cells. Thus mannan is an
inhibitor of macrophage as well as neutrophil candidacidal activity.

The mechanisms by which macrophages and monocytes kill Q. albicans
are not well understood. Lehrer (1975) described both a myeloperoxidase
dependent and independent mechanisms of candidacidal activity for mono-
cytes. Patterson-Delafield _t _L. (1980) described cationic proteins
from alveolar macrophages as having candidacidal activity. Sasada and
Johnson (1980) has suggested that superoxide is a major mechansim by
which macrophages kill Q. albicans. Mannan has been shown to interfere
with myeloperoxidase both in neutrophils and in a cell free system by
Wright gt_al,, (1983). However, mature macrophages lack myeloperoxidase
(Murray, 1984) so another mechanism(s) must come into play. The pres-
ence of superoxide in macrophages exposed to mannan was measured by
nitroblue tetrazolium blue reduction (Figure 16). Mannan was shown to
decrease the amount of superoxide in macrophages regardless of the
opsonization of the yeast cells.The decrease in superoxide of the macro-
phages could be accomplished by two means: a decrease in the oxygen
uptake of the macrophages or by the dismutation of superoxide into
hydrogen peroxide. Nelson _t _L. (1984) demonstrated that mannan has a
superoxide dismutase like activity.

Another level that mannan may interfere with the candidacidal
activity of macrophages is by inhibiting phagolysosome fusion. There

were several reasons for pursuing this mechanism. First, D'Arcy Hart

(1981) demonstrated that phagolysosome fusion is necessary for the

 

76

killing of S. cerevisiae by macrophages. Second, the presence of

 

cationic proteins as a potent microbicidal substance and in the absence
of oxidative metabolism may provide a secondary line of defense for the
host. Finally, in_ vit£g_fusion of neutrophil phagocytic vesicles and
lysosomes could be inhibited by the presence of mannose (Amano and
Mizuno, 1981). Since it is possible that mannan may be internalized by
the mannosyl/fucosyl glycoprotein receptor and that the intracellular
fate of mannose terminal glycoproteins is not known it is possible that
these glycoproteins may be degraded and released into the cell or in
some way interfere with phagolysosome fusion. The results obtained in
this study (Figure 17) indicate that mannan does not interfere with the
phagolysosome fusion. This indicates that in Con A peritoneal
macrophages that mannan interferes with superoxide and that lysosomal
contents plays little or no role in the intracellular killing of Q.
albicans.

The results of this study indiate that mannan is capbale of af-
fecting the ability of macrophages to phagocytize Q. albicans via the
mannosyl/fucosyl glycoprotein receptor and the complement receptor,
probably the CR3, which may be an important pathogenic mechanism in
patients with low levels of immunoglobulins. Also, mannan might influ-
ence pathogenicity because it diminishes the amount of superoxide
present in macrophages thus reducing the ability of macrophages to kill
the ingested yeast cells. It is unlikely that phagocytic cells are
exposed to such high concentrations of mannan in the host with the
exception of sites of infection or in patients with chronic mucocuta-

neous candidiasis where serum mannan level of 2.7 mg/ml have been re-

 

77

ported (Wright gt aL., 1981). It is important to note that mannan does
not inhibit the phagolysosome fusion within macrophages thus inhibition
of this fusion is not an aid in establishing an infection in candidi-
asis. An avenue of investigation that may help in the understanding of
how mannan may be an aid in establishing an infection is to examine the
effects of long term exposure of low doses of mannan on lymphocytes and

phagocytic cells, especially macrophages.

 

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