STUDEES ON A POSSiBLE CELLULAR RESPONSE RN
MICE {MMUNIZED WITH STAPHYLOOOCCUS AUREUS,
SMITH STRAIN DIFFUSE

Thesis for the Degree of M. S.
Micmem STATE UWERSITY
DONNA Y. MUERHEAD
1970

 

 

 

 

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STUDIES ON A POSSIBLE CELLULAR RESPONSE IN MICE

IMMUNIZED WITH STAPHYLOCOCCUS AUREUS, SMITH STRAIN DIFFUSE

 

By

1

Donna Y; Muirhead

A THESIS

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

MASTER OF SCIENCE
Department of'Microbiology and Public Health

1970

CG7‘7/4/

ABSTRACT

STUDIES ON A POSSIPLE CELLULAR RESPONSE IN MICE
IMMUNIZED WITH STAPHYLOCOCCUS AUREWS, SWITH STRAIN DIEEUSE

 

by

Donna Y. Muirhead

Intracellular inactivation of Staphylococcus aureus,

 

Smith strain diffuse, by macrophages of unimmunized mice and mice
immunized with dead, or live staphylococci, or ECG was impossible
to determine without the addition of some antibiotic. Washing the
parasitized macrophages did not reduce the extracellular population
of staphylococci sufficiently to follow rates of intracellular
killing: the extracellular population multiplied to such an extent,
no differences could be detected. The addition of a small (3 ug/ml)
amount of dihydrostrentomycin allowed differences to be determined.
Macrophages from BCG immunized mice inactivated staphylococci to a
measureable extent compared to macronhages from control, dead or live
staphylococci immunized mice. No significant differences were observed
among these latter groups.

At pH 5 bactericidal activity of macrophage lysates was
detected for all test groups against both Opsonized and unopsonized

staphylococci. Inactivation of unopsonized staphylococci was observed

at higher lysate dilutions than Opsonized staphylococci. Lysosomal
acid hydrolases were implicated because of their acid pH optima. No
bactericidal activity was seen for any of the four experimental groups
at pH 5 or 7 in the presence of 5% hom010gous mouse Serum. In the
absence of mouse serum, staphylococcal inactivation at pH 7 was ob-
served only with lysates from BCG immunized mice. A rough fraction-
ation of lysate from this positive control group indicated a lyso-
somal origin for this activitv. No tests were made to determine the
cellular origin of this factor(s).

An increased percentage of mice immunized with live staphy-
lococci survived an intraperitoneal challenge dose of 100 LDSO
staphylococci. On the other hand, mice immunized with ECG or dead

staphylococci were protected to some extent, but none of the control

mice survived challenge.

ACKNOWLEDGEHENTS

The author expresses gratitude to Dr. C. L. San Clemente for
his guidance and assistance throughout the course of this study.

Thanks are also extended to Miss Aline Garretson and Mrs.
Doris Beck for their useful suggestions and encouragement which

aided in the completion of this thesis. '

ii

TABLE OF CONTENTS

LISTOFTABLES o o o 0 O O o o o o o e o o 0

LIST OF FIGURE 0 C O O O O O O O O O O O O 0

INTRODUCTION

LITERATURE REVIEW 0 O O O O O O O O O O O O 0

Cellular immunity . . . . . . .

Staphylococcal pathology and immunology

Immunological characteristics .
Metabolic characteristics . .

Survival within host tissues . _
Immunology . . . . . . .

\

l

“mum AND HEPHODS O O O O O O ,0 O O O O O O

Immunization . . . . '. . . . . .

Animals . . . .. . . . . .
Bacterial strains . . . . . .
Test media for bactericidal assays
Bacterial media . . . . . .
vaccines and immunization schedules
Bleeding procedure . . . . . .
Opsonization of staphylococci . . .
Prestimulation and collection of peritoneal
macrophage exudates . . . . . . . .
Determination of LDSO for Staphylococcus aureus

0 o 0 e 0 I o
O o O o o o 0
e O o o e o O O
C O O O O O 0 O

Bactericidal assays . . . . . . . . . .

MacrOphage lysis with delta-hemolysin . . .
Rate of intracellular killing . . . . . .
Effect of washing macrophages on extracellular
growth of staphylococci . . . . . . .
Staphylococcal growth curves in the presence
of dihydrostreptomycin . . . . . .

.Assay for bactericidal activity of macrophage

lys ates o o o o o o e o - ‘ e O O
Fractionation of macrophage lysate for
localization of bactericidal activity . .

iii

0 O O O O O O O O O O O 0 O

O O O Q Q

SODCDQWU u H 3.

5

assesses

RES” LTS 0 e o o o o o ' e o o a e o o o o o o

Extracellular growth of staphylococci . . . . .
mSO of Staphlecoccus aureus in mice via
peritoneal route . . . . . . . . .
Staphylococcal growth in suspension with

peritoneal macrophages . . . . ' . ..

Extracellular growth of staphylococci after
repeated washing of parasitized peritoneal
macrophages . . _. . . . . . . . .

Staphylococcal growth in presence of different
amounts of dihydrostreptomycin . . . . .

Bactericidal activity of peritoneal macrophages and'
peritoneal macrOphage lysates .. . . . . . .

Intracellular inactivation of staphylococci by
washed mouse peritoneal macrOphages in
suspension with 3 ug/ml dihydrostreptomycin

‘Bactericidal'activity of diluted macrophage "
lysates against staphylococci . . . . .

Localization of bactericidal activity in
macrophage lysates from BCG immunized mice .

Effect of staphylococcal challenge on survival
of immunized mice . . . . . . . . .

DISCUSSION 0 O O O ‘ O O O O O O O O O O O O 0

Cellular immunity and the problem of antibiotics . .
Bactericidal activity of immune versus normal

. .macrophages .. ., ....- . '., . .. <.. . . ..,I,

SMIARY............'.......
ADDENDUM.¢..............A.

LITERATURECITED, . . . . . . . . . . .

iv

Page
22«
22‘-

.22

22

28
28

2e
31-
to
b1
tn 1

- L5
be
be
SO

. Table

1.

7.

LIST OF TABLES

 

The LDS for Staphylococcus aureus with female
Swiss aIbino mice via the peritoneal route . . . .

' Effect of serial 3-fold dilutions of macrophage lysate

on staphylococcal viability. Test medium: 0.0714
citrate-phosphate buffer, 0.01% BSA, 5% mouse serum

at pH 9.0 o o e o e e e o e e e o o 0 .°.

Effect of serial 3-fold dilutions of macrOphage lysate
on staphylococcal viability. Test medium: 0.0714.
citrate-phOSphate buffer, 0.01% BSA, 5% mouse serum

atpH7. o o o e e or. o e o o o o o 0'

Effect of serial 3-fold dilutions of macrophage lysate
on viability of epsonized staphylococci. Test medium:
0.07lfl citrate-phosphate buffer.and 0.01% BSA at pH 5.

Effect of serial 3-fold dilutions of macrophage lysate
on viabiltiy of opsonized staphylococci. Test medium:
0.0? M citrate-phosphate buffer and 0.01% BSA at pH 7.

"~Effect of serial 3-fold dilutions of macrophage lysate

on viability of staphylococci. Test medium: 0.0? M
citrate-phOSphate buffer and 0.01% BSA at pH 5.. .. .

Effect of serial 3-fold dilutions of macrophage lysate

'on staphylococcal viability.’ Test medium: 0.0? M

citrate-phOSphate buffer and 0.01% BSA at pH 7.. . .

Effect on staphylococcal viability of serial 3-fold
dilutions of fractions from macrOphage lysate obtained

by differential centrifugation. Source of macrophages:

BOG immunized mice. ‘Medium: 0.0714 citrate-phOSphate
buffer and 0.01% BSA at pH 7.. . . . . . . . .

Effect of a challenge dose of staphylococci on‘the

'survival of test miCe . .. . . . . . . . . .

A"

'Page

. 23.

3h

35
36
37
38 .

39

h2

h3

LIST OF FIGURES

Figure

3.

Total staphylococci, both extracellular an macrophage
associated, vs. time in the presence of 10 peritoneal
large mononuclear cells/ml and without streptomycin .

Extracellular popu ation of staphylococci vs. time in
the presence of 10 large mononuclear cells/ml and
without streptomycin. . . . . . . . . . . .

Rate of killing of staphylococci by peritoneal mouse
macrOphages following repeated washings of macrophages
after incubation with staphylococci for 15 minutes at
37 C and without streptomycin . . .- . . . . .

Staphylococcal growth curve in the presence of different
amounts of streptomycin _. . ._ . . . . . .1}~

Extragellular staphylococcal growth curve in the presence

of 1 mouse peritoneal macrOphages/ml and different
mounts Of StreptWYCin- _. o e e e o e e e, 0

Rate of intracellular killing of staphylococci by

peritoneal mouse macrophages in the presence of 3 ug/ml
streptomycin following repeated washings of macrophages

after incubation with staphylococci for 15 minutes
‘t37c'~. . O O O O 0' O C O O C O C C 0

e1

Page

2h

25

27

29

30

33

INTRODUCTION

Cellular immunity, an immunological state associated with an
increased bactericidal activity of macrOphages, is generally induced
only with viable absolute or facultative intracellular parasites. The
organisms associated with the induction of this phenomenon are phylo-
genetically diverse: viruses, bacteria and protozoa all appear as
suitable stimuli (Ruskin et al., 1969). Although the response appears

to be spcifically elicited by intracellular parasites, the effect is

 

nonSpecific in nature, i.e., immunization with Mycobacterium tubercu-
lggig induces a resistance against other infectious agents (Mackaness,
196h; Coppel and Youmans, 1969A, 1969B; Dubos and Schaedler, 1957;
Youmans and Youmans, 1969).

Staphylococcus aureus is generally considered an extra-

 

cellular parasite: however small numbers persist within macrophages
and are inactivated only after an extended period of time (Kapral
and Shayegani, 1959: Mackaness, l96h). A possible cellular in-
volvement has been suggested for staphylococci on the basis of this
slow degradation (Shayegani, 1968: Shayegani and Mudd, 1966).
Staphylococcal infections are often associated with delayed-type
hypersensitivity, an immunological state associated with cellular
immunity (Mackaness, 1967: Taubler, 1968: Taubler and Mudd, 1968).

Most studies of cellular immunity are made by measuring

f'u

rates of bactericidal activity against homologous and heterologous
organisms. .12.!339 testing most often involves counts of infectious
units over time in specific body tissues such as Spleen, liver and
blood Oiackaness, 196h: Blanden et al., 1969). 13.33359 testing
involves measuring the rate of intracellular killing within macro-
phages in monolayers (Ruskin et al., 1969zliiller and Twohy, 1969)
or in suspension (COppel and Youmans, 1969A, 1969B). lg 31259
studies invariably incorporate the use of antibiotics to remove the
extracellular pOpulation. Bonventre and Imhoff (1970) find dihydro-
streptomycin has a bactericidal effect on intracellular mycobacteria.
0n the basis of these observations we undertook a study to
test for a possible cellular reSponse in mice to injections of staph-
ylococci. Our experiments were designed: (i) to determine the rates
of intracellular inactivation by mouse peritoneal macrOphages with
and without the addition of dihydrostreptomycin, and (ii) to measure
the bactericidal activities against staphylococci by macrophage
lysates from mice immunized with staphylococci as compared with

positive and negative control groups.

LITERATURE REVIEW

Cellular immunity

 

Immunological characteristics.--The ability to ingest and

 

destroy invading microorganisms is a primary defense mechanism in
animal systems. Occasionallyt bacteria invade a host and are phago-
cytosed by macrophages but manage to resist intracellular destruction
and even reproduce themselves intracellularly. The animal host
eventually counters this challenge by producing "resistant macro-
phages" (Melly et al., 1960), highly efficient cells which are able
to ingest and destroy the established organism. At this point,
the animal is said to have developed a "cellular immunity" (Mackaness,
1961:). i

It has been shown (Mackaness, 196h: Coppel and Youmans,
1969A, 1969B: Dubos and Schaedler, 1957: Youmans and Youmans,
1969) that immunization with facultative intracellular parasites
induces an immunity not only against the homologous immunizing
organism, but also against heterologous facultative intracellular
organisms and organisms normally considered to be extracellular,

i.e., immunization withliycobacterium tuberculosis produces an

 

immunity which not only protects against mycobacterium but also

against listeria, brucella and staphylococcus. This resistance

may extend to phylogenetically diverse organisms (Ruskin.et al.,

1969). Resistance to Toxoplasma gondii produces a cross protection

 

against listeria, salmonella, mengo virus and besnoitia.

The phenomenon is not completely nonspecific, however, for
it has been shown that the recall of the response is elicited only
by the original immunizing organism. Although the resistance is
nonspecific in its action, the state of cellular resistance is de-
finitely related to the immune mechanisms because (i) the cells
require a certain period of time to respond, (ii) immune animals
slowly decrease their resistance but regain it quickly upon rechal-
lenge with the immunizing organism, and (iii) resistance can be
enhanced by additional injections with the same organism (Mackaness,
1967). An animal host which has develOped a cellular immunity may
eliminate both homologous and heterologous challenge organisms:

_ however, the immunity appears much more efficient against the homo~
logous infection (Ruskin et al., 1969).'

Macrophages can be "activated" by many different types of
irritants and toxins such as tuberculin, endotoxins, zymosan,
colloids and hormones (Nelson, 1969). Injection of animals with
these substances produces changes in morphological pronerties and
activities of macrophages similar to those seen in immune cells.
Responses due to nonSpecific irritation and those of a purely
immunological nature are difficult to separate although attempts
were made by Coppel and Youmans (19698, 19690) and Youmans and
Youmans (1969). These investigators observed that immunization

with viable fl, tuberculosis was at least several hundred times

 

'lore effective than killed cells in challenge against tuberculous
infections. This superiority was not seen in mice challenged with

Listeria monocytoeenes or Klebsiella pggumoniae. They found that

 

 

living and heat killed mycobacterial cells induced protection
equally well against the heterologous organisms and suggested
there may'be at least two factors in mycobacterial cells important
in immunization. They proposed that one might be a fairly stable
ce11.wall component which acts similar to the manner of E. 321}
endotoxin and the other a thermolabile material which induced
inunological protection only against 14.. tuberculosis. Hackaness
(1967) suggested that the defense mechanisms against a homologous
challenge were related closely with the development of delayed
hypersensitivity, an immunologic phenomenon with a high degree of
specificity.

.ff, Cellular immunity is associated with a form of resistance
which cannot be transferred with immune serum, but only with cells
of the reticuloendothelial system (Maokaness, l96h: Jenkin and
gowley,‘l963). Transfer of an immunity to an intracellular para-
site may'be accomplished by lymphoid cells (mediators of delayed
hypersensitivity) derived only from mice infected with that
particular organism (Mackaness, 1968). Although the donor mice
show resistance to heterologous bacterial challenge, mice which
had received immune lymphoid cells manifested a resistance only
against the homologous organism.‘ Similar results were shown

for facultative intracellular protozoans such as toxoplasma and
besnoitia (Frenkel, 196lli: Normal mice receiving lymphoid cells
from.toxop1asma immune animals were resistant only to toxoplasma

and normal mice receiving lymphoid cells from besnoitia immune

animals were resistant only to besnoitia.

‘Most of the in 31159 studies made on the intracellular degra-
dation of s+aphylococci involved the use of antibiotic (usually dihy-
drostreptomycin and/or penicillin) in the test medium, a questionable
practice in light of recent reports showing uptake of dihydrostrepto-
mycin (Bonventre and Imhoff, 1970) and a bactericidal effect on intra-
cellular mycobacterium (Patterson and Youmans, 1969). Patterson and
Youmans observed no difference between normal and "immune" macrophages
in their ability to inactivate intracellular mycobacterium in the
absence of antibiotic, but saw only an increased survival rate of
"immune" macrophages. They did observe increased bactericidal activity
within "immune" macrophages in the presence of antibiotic and suggested
this effect was caused by a greater permeability or uptake of dihydro-
streptomycin by these cells as compared with normal cells. The rate
of inactivation was proportional to the amount of dihydrostreptomycin
added to the medium and they observed an effect with as little as
1.25 ug/ml. Bonventre and Imhoff (1970) observed that macrophages
actually accumulated tritiated dihydrostreptomycin intracellularly to
higher concentrations than were found in the extracellular milieu,
although the rate of uptake was extremely slow in comparison to other
substances which were transported across membranes. They reported
concentrations only in terms 6r radioactive activity and did not relate
this on a weight basis, therefore no direct comparisons could be made

with other results. In in vitro tests using Mycobacterium tuberculosis

 

sixteen times the concentration of antibiotic was required to in-
hibit the growth of intracellular bacilli than was needed to inhibit
growth in culture medium Oiackaness, 1952). Deepite this evi-

dence which perhaps argued against a cellular mechanism, not all

work was done with jg vitro systems using antibiotics. lg vivo experi-
ments prove this response was real and was not an artifact (Mackaness,
1961;: Blanden et al., 1966, 1969: Dubos and Schaedler, 1957).

Metabolic characteristics.«Macrophages from immune animals

 

with increased ability to inhibit the growth of tubercle bacilli in-
gested carbon, tubercle bacilli and staphylococci more readily than did
macrophages from normal animals (Lurie, 1939) and had an accelerated
mitotic activity (Khoo and Mackaness, 1961;: Mackaness, 1962). Hyrvik
66:1. (1962) and Evans and Myrvik (1967) noted enhanced metabolic)
activities by immune alveolar macrOphages such as increases in oxygen
uptake and a heightened response in the hexose monophosphate shunt.
Increases in glycolytic activity of peritoneal macrophages as well as
increases in acid hydrolases were associated with the cellular immune
state (Saito and Suter, 1965). Cohn and Wiener (1963) found increased
levels of acid phosphatase, lipase and lysozyme in BOG-induced alveolar
macrOphages as compared with control cells. Mouse peritoneal macro-

phages imune to Corynebacterium‘ovis had a total cell protein 1.85

 

times that of normal phagocytes (Hard, 1970). Substantially higher
activities of acid phosphatase, 5-g1ucuronidase, cathepsin D, lysozyme,
N-finzoyl-DL-phenylalanine-l-naptho1esterase, 9-napthy1acetate esterase
’ and aryl sulfatase were observed in corynebacterium-imune macrophages 6
as compared to control cells. Dannenberg and Bennett (1963), however,
found no increase in levels of proteinase, esterase or lipase of
immune peritoneal mononuclear cells from mycobacterium infected animals.
" 1n spite of the mass of data on this subject the exact nature and
the mechanistic‘details of this phenomenon remain to be clarified.
Similarly, its relationship to other immunological reaponses such as

humeral antibody production and delayed hypersensitivity is unclear.

Staphylococcal pathology_and immunology

Survival within host tissues.-—Staphylococcus aureus is

 

generally considered an extracellular parasite although Melly et a1.
(1960) and Hunt and Moses (1958) reported not only intracellular
survival, but actual multiplication within human and mouse leucocytes.
Kapral and Shayegani (1959) noted virulent staphylococci survived
but did not multiply in leucocytes of normal rabbits and humans.
Haokaness (l96h) found staphylococci more rapidly inactivated within
granulocytes than within.macrophages, a view in accord with Ketch-
nikoff's morphological studies (Hirsch, 1959). Mackaness reported
'less than 0.1% staphylococci surviving within granulocytes after

an incubation period of three hours. This survival rate contrasted
with 3 to 10% in the case of macrophages. Long term studies of this
small surviving population of staphylococci have not been made,
primarily because of the difficulties involved in eliminating the
extracellularwmicrobial population.

' The virulence of pathogenic staphylococci has been attri-
buted to many factors. Breakdown in natural defenses and genetic
defects within the host as well as environmental stress have been
implicated in infectious events (Florman, 1968: Horse, 1968: Zeya
and Spitznagel, 1968). Tauraso and White (1963) cited a variety of
toxins produced by virulent staphylococci such as alpha hemolysin,
dermonecrotoxin and leukocidin. The presence of antiphagoqytic

agents was also found to be contributory to the disease state.

Blair (1965)-observed staphylococcal leucocidin had a unique cyto-

9

toxic effect on white blood cells. Virulent staphylococci were
generally associated with coagulase and other plasma coagulating
factors that were secreted or retained at the cell surface to
produce aggregates of infecting cells that were phagocytosed with
difficulty (Smith, 1963).

The encapsulated Smith strain of Staphylococcus aureus was

 

isolated from a patient with osteomyelitis in 1930 and has shown

a consistent pathogenicity for mice. The diffuse and compact

variants were distinguished by their colonial morphology in serum-

soft agar (Yoshida and Ekstedt, 1968). The extreme virulence of the
Smith diffuse variant has been attributed to some component of its
antiphagocytic capsule. The Smith compact variant was avirulent

and was rapidly ingested by peritoneal phagocytes. Both variants
elaborated free coagulase and alpha hemolysin (Koenig, 1962: Koenig

et al., 1962 A, 1962 B: Parker et al., 1965: Hunt and Moses, 1958).
Rogers (1962) suggested these strains might be indicators of char-
acteristics acquired by other pathogenic staphylococci in 32.2332
multiplication which were lost 39 31333. Three lines of evidence

were prOposed which might support such a thesis: (1) virtually all
adult human sera had detectableOpsonizing antibody against the.

Smith diffuse variant suggesting the antigen to be fairly common:

(ii) there was evidence of antigenic relationships between the Smith
diffuse strain and pathogenic staphylococci from human infections as
noted when immunization with these strains induced production of a heat-
stable antibody which could opsonize the Smith strain: and (iii) there
were bacterial precedents to support lg 1:39 changes in bacterial

characteristics. Certain strains of Pasturella pgstis encapsulate

 

and acquire resistance to phagocytosis $2 vivo.

10

No one factor has been consistently associated with viru-
lence. IHorse (1968) suggested all are important and the more
virulence factors a strain possesses the greater are its chances for
establishing infection.

Immunology.--Increased resistance to challenging doses of

 

Ԥ. aureus measured by survival tests was observed after immunization
with specific antigens and both live and dead vaccines (Ekstedt,
1965). Hyperimmune rabbit antisera against heat-killed vaccines

of the Smith diffuse strain of S. aureus protected mice against

 

challenge with the homologous organisms (Ioshida and Ekstedt, 1968).
Absorption of this sera with purified teichoic acid removed the
protective antibody. Immunization with specific antigens such as
coagulase, alpha hemolysin, cell wall teichoic acids and protein A
protected animals to some extent, but, in general,;vaccination'against
staphylococci has met with little success (Rogers and Melly, 1965:
Koenig and Kelly, 1965: Lominski et al., 1962; Harrison, 1961;:
Spencer et al., l96h: Ekstedt and Yoshida, 1969).

Johnson et al.,(1960) suggested staphylococci closely
resembled the tubercle bacillus in its ability to maintain a long
term residence within host tissues and survive within phagocytic
cells. Rogers and _Helly (1965) admitted intracellular residence
might be an.important attribute of virulent strains, but maintained

“vextracellular’multiplication was also of prime significance.

Goshi et a1. (1961) noted repeated intradermal infection

- a —

of rabbits could result in an increase in susceptibility to infec-

tion rather than an increase in resistance. This was associated I“
with the development of delayed hypersensitivity to the staphylo-

cocci.

ll

Malata et a1. (1969) found 16% of healthy persons showed
immediate and 18% showed delayed hypersensitivity to an antigenic com-
plex of ten different staphylococcal strains. Eighty-eight percent of
patients with furunculosis produced a positive immediate reaction
to this complex, while 66% showed positive reactions of the delayed
type. They prOposed that an effective therapy would probably not
be based on antibody production. Greenherg (1968) and Spencer et a1.
(l96h) observed persons with persistent and recurring infections
had relatively lfixfil titers of both alpha antitoxin and antileuko-
cidin and reasoned there was little value in an attempt to increase
these titers through immunization. Taubler (1968) and Taubler and
Mudd (1968) have demonstrated delaved-type hynersensitibity to a

nonencansulated strain of Staphylococcus aureus by in vivo (footpad)

 

and E m (migration inhibition) tests.

Although delayed-type hypersensitivity and cellular immunity
develop almost simultaneously with mycobacterium infected animals,
different components of the tubercle bacillus might cause the dif-
ferent reSponses. The protein (tuberculin) might be responsible for
the delayed hypersensitivity and the lipids or polysaccharides or
both may be responsible for the immunity (Dannenberg, 1968). Mack-
aness (1967) conceded that it is possible to have delayed hyper-
sensitivity without cellular immunity and vice versa. A cellular
immunity without delayed hypersensitivity may be achieved by
desensitization with antigen. However, he does suvwest a strong
correlation between the two responses.

The surface antigen of the encapsulated Smith diffuse strain
of S. EEIEEE has been isolated, purified and characterized byliorse

(1962). The antigen contained 701 carbohydrate. Rabbits immunized

12

with heat-killed cells of the Smith strain exhibited cutaneous
hypersensitivity when small quantities of Smith surface antigen
(SSA) were inoculated intradermalky. Intracntaneous inoculation

of 100 ug of SSA into normal rabbits produced no local reaction,
whereas as little as 1 ug injected into immunized animals produced
an erythema and swelling 1 to 2 centimeters in diameter. These
reactions were maximal at 8 to 12 hours (immediate-type hypersensi-
tivity) and gradually receded over the next hB hours.

Shayegani (1968) and Shayegani andliudd (1966) suggested
a.possible cellular involvement for staphylococci by virtue of the
slow degradation process. They proposed that "these facultatively
intracellular parasites" might outlive the monocytes in which they
reside and by multiplying extracellularly produce constant, recurring
infections. 1

MATERIAIS AND METHODS

Immunization

 

Animals. Female Swiss Albino mice (Spartan Research Ani-
mals, Haslett, Michigan) weighing 2b to 26 grams were used in all
experiments. They were housed h to 5 per cage. Food (Purina
mouse chow, Ralston Purina Co., St. Louis, Mo.) and water were given
ad; libitum.

Bacterial strains. Staphylococcus aureus, Smith diffuse

 

 

strain was donated by Dr. Joseph J. Kowalski, Dept of Microbiology

and Public Health, Michigan State University. Hycobacteriml tuber-

 

 

culosis, Bacille Calmette-Guerin was obtained from Dr. Virginia
Hellman, Dept. of Microbiology and Public Health, Michigan State
University.

Bacterial media. Modified Staphylococcal 110 medium was pre-

 

pared by the method of Yoshida and Ekatedt (1968). Dubos broth (Difco
laboratories, Inc., Detroit, Mich.) supplemented with 0.5% dextrose
was used to grow mycobacterium.

Test media for bactericidal assays. Measurements of intra-

 

cellular populations of staphylococci in the presence, of mouse peri-
toneal macrophages were made using 90% Hank's Basal Salt Solution
(HBSS)(Microbiological Associates, Inc., Bethesda, Md.), 10% mouse
serum and 0.0056 M glucose.

Measurements of staphylococcal growth in different concentra-

tions of dihydrostreptomycin with and without the presence of mouse

p

1h

peritoneal macrophages were made using 90% H835, 10% Fetal Bovine
Serum (FPS)(Microbiological Associates, Inc., Bethesda, Md.) and
0.0056 M glucose to conserve mouse serum.

Vaccines and immunization schedules. Four groups of test mice

 

were used: (i) control mice, (ii) mice immunized with dead staphylo-
cocci, (iii) mice immunized with live staphylococci, and (iv) mice
immunized with live mycobacteria.

Control mice were not immunized. A killed staphylococcal
vaccine was prepared using an 18 hour culture of §. aureus incubated
at 37 C. The cells were centrifuged and resuspended in culture super-
natant to 108 CFU/ml as determined by Optical density readings at
620 nm. Ten milliliter aliquots of the susnension were transferred to
sterile vaccine bottles and benzalkonium chloride was added to a con-
centration of 1:1600 (v/v). The suspension was shaken vigorously and
placed in a 37 C water bath for 30 minutes. The vaccine was subse-
quently tested for sterility by inoculating into tubes containing
Brain Heart Infusion Broth (Difco Laboratories, Detroit, Mich.).
These tubes were then incubated at 37 C for 3 days and observed for
growth. Mice were immunized on a biweekly basis for 1 month by
injecting 0.1 ml of this vaccine (107 killed organisms) intraperitoneally.
Mice immunized with live staphylococci were treated in a manner similar
to the dead staphylococci~treated mice. On the day of immunization
an 18 hour culture was centrifuged and resusnended in sterile 0.85%
saline to 108 CFU/ml as calculated from previous measurements of
viable staphylococci versus optical density readings (Bausch and Lomb
Spectronic 20) at 620 nm. In addition to biweekly intraneritoneal
injections of 107 CFU, an open, pustular lesion was induced 2 weeks
prior to testing by injecting 0.1 ml of the live vaccine subcutaneously

on the abdomen. (These healed in 3 to 8 days depending on the severity

15

of the lesion.) Mice immunized with mycobacteria were injected once
intraperitoneally h weeks prior to testing with 0.2 ml of a saline
suspension of a 10 day culture incubated at 37 C. The cells were
suspended to an Optical density reading of 0.15 at 690 nm which repre-
sented 108 cells/ml. The mice were therefroe injected with approxi-
mately 2 x 107 viable cells.

Bleeding procedure. Two days following a challenge immuni-

 

zation, blood samples were obtained via the retro-orbital plexus.
Approximately 0.5 to 0.75 ml of whole blood was taken per mouse and
about u ml of serum was obtained per 20 mice. Blood was taken from
the mice not more often than once a month. Pooled serum from each
group was frozen at -20 C until used.

Opsonization 2f staphylococci. A pellet of cells obtained

 

 

by centrifuging 1 ml of an 18 hour culture (approximately 109 CFU)
was suSpended in 0.3 ml of the appropriate mouse serum. The cell-
serum mixture was incubated at room temperature for 20 minutes. The
staphylococci were then centrifuged and the serum removed. The cell
pellet was resusnended and diluted in saline to a calculated 2 x 103
CFU/ml and used immediately in the bactericidal assay with macrophage
lysates.

Prestimulation and collection 23 peritoneal macrophage exudates.

 

 

All work was performed with macrOphages of peritoneal origin. Two days
prior to testing, mice were injected intraperitoneally with an im-
munizing dose of the appropriate vaccine and 1 ml of sterile 0.1%
glycogen (Nutritional Biochemicals Corporation, Cleveland, Ohio).
Control mice received glyc0gen only. On the day of testing, 2.5 ml

of sterile HESS containing 10 units/ml sodium heparin (Wolins Phar-
maceutical, Inc., Farmingdale, N. Y.) was injected into the peritoneal

cavity. The abdomen was then massaged by gentle kneading for a few

16

minutes. The peritoneal exudate was collected with a hypodermic
syringe equipped with a special 22-guage stainless steel needle per-

forated at the needle tip with 3 to h small holes.

 

 

Determination Ef-LQSO for Staphylococcus aureus. An overnight
culture of staphylococci was centrifuged and resuspended in sterile
0.85% saline. Five-fold dilutions of the cell suspension were made
in saline and 1.0 ml volumes were injected intraperitoneally.

Mortality rates were based on the number of mice dead by 20 hours.

Bactericidal assays

 

'Macrophage lysis by delta-hemolysin. Delta-hemolysin (supplied

 

 

by Dr. Frank A. Kapral, Ohio State University) is an exotoxin produced
by staphylococcus which lyses erythrocytes and leucocytes. A concen-

tration of 25 to 50 HDS units/ml was suggested for lysing mouse peri-

0
t0neal.macr0phages (Dr. Frank Kapral, personal communication). Prelim-
inary testing indicated that 95% or better of 106 large mononuclear
cells/ml were lysed after 15 minutes incubation at ambient temperature
with 50 HDSO units. This concentration was used in all experiments

requiring lysis of mouse peritoneal cells.

Rate gf intracellular killing. Mice were prestimulated 2 days

 

prior to testing with an immunizing dose of antigen and 1 ml of sterile
0.1% glycogen as described above. On the day of the test macrophages
were collected, washed in H888 and resuSpended in media consisting of
90% HBSS, 10% mouse serum and 0.005614 glucose at pH 6.8. The cells
were counted with a haemocytometer (American Standard Haemocytometer,
Arthur H. Thomas Co., Philadelphia, Pa.) and suSpended in test medium.

6

to 10 cells/ml. An 18 hour culture of S. aureus was centrifuged and

resuspended in saline to a calculated concentration of 109 CFU/ml.

17

One-tenth milliliter of the staphylococcal suSpension was added to

10 ml of the macrophage suspension for a final staphylococcuszmacrOphage
ratio of 10:1. The cell suspensions were incubated at 37 C on a gyro-
rotatory water bath (New Brunswick Scientific Co., In., New Brunswick,
N. J .). One milliliter samples were taken at appropriate times over a
11 hour period and transferred to conical centrifuge tubes. The sus-
pensions were centrifuged at 500 x g for 5 minutes (International
Centrifuge, Model SBV,‘ International Equipment Co., Boston, Mass. ).
The'supernatant fluid was carefully removed with a sterile Pasteur
pipette and transferred for serial dilution and plating on nutrient
agar to provide the extracellular count. Additional 1 ml samples of
the 10:1 suspension were removed and mixed with 0.1 m1 of 500 HDSO
units/m1 of delta-hemolysin. After incubating for 15 minutes at room
temperature the macrOphage lysate was briskly pipetted up and down for
one minute before dilution and plating on nutrient agar. This latter
procedure provided the total staphylococcal pOpulation, i.e., both
intracellular and extracellular counts.

. , In subsequent experiments to determine the rate of intracellular
inactivation, macrOphages were collected, washed with HBSS and 2 x 107 .
cells were resuspended in 1.0 m1 of test medium consisting of 90% HBSS,
10% mouse serum and 0.0056 M glucose. An 18 hour culture of S. m
was centrifuged and resuspended in saline to 2 x 109 CPU/ml. One-tenth
milliliter of the staphylococcal suspension was added to the macrophage
suspension to obtain a staphylococcuszmacrOphage ratio of 10:1. The
suspension was incubated at 37 C for 15 minutes on a gyrotatory water
bath, after which the cell suspension was centrifuged and washed twice

with 30 ml saline. All centrifugation was done at low gravity forces

18

(500 x'g for 5 minutes) to sediment the macrOphages but leave the
staphylococci in the supernatant fluid. The macrophage pellet was
then resuspended in 1.05 ml of culture medium. One-half milliliter
was transferred to an additional 9.5 ml of culture medium containing

3 ug/ml of dihydrostreptomycin (Pfizer Laboratories, New York, N. Y.)
and 0.5 m1 transferred to 9.5 m1 of culture medium without dihydro-
streptomycin. One milliliter of test suspension was removed from

each system at appropriate time intervals over a h hour period. The
macrophages were centrifuged, resuspended in 1 m1 of 50 HDSO units/m1
of delta hemolysin and incubated at ambient temperature for 15 minutes.
The macrOphage lysate was then serially diluted and plated on nutrient
agar to obtain the concentration of intracellular staphylococci.

Effect 2; washing macrOphages 29 extracellular growth 2f staph-

 

 

ylococci. Peritoneal macrOphages were collected, washed, counted
and resuspended in 1.0 m1 of test medium (consisting of 90% HBSS, 10%
mouse serum and 0.0056 M glucose for a final concentration of 107
large mononuclear cells/ml. One-tenth milliliter of an 18 hour
culture of §..§ggggs, centrifuged and resuspended in saline to 109
CPU/ml, was added to the macrOphage suspension for a staphylococci:
large mononuclear cell ratio of 10:1. The cell suspension was incu-
bated for 15 minutes at 37 C before centrifugation and washed twice
with 30 m1 of saline. After the last wash the macrOphages were resus-
pended in a small volume of test medium and transferred to a sterile
flask. Additional test medium was added for a final concentration
of 106 large mononuclear cells/m1. One milliliter was immediately
removed for a O-time reading, transferred to a conical centrifuge
tube and centrifuged. The supernatant fluid was carefully removed

from the cell pellet with a sterile Pasteur pipette and the cell

l9

pellet was resuspended in 1 ml of 50 H050 units/ml of delta hemolysin.
After incubation in delta hemolysin for 15 minutes at room temperature
the cell lysate was'briskly pipetted for a minute before serial dilu-
tion and plating.

Staphylococcal growth curves E the presence of dihydrostrepto-

 

 

m. An 18 hour culture of staphylococci was centrifuged and resus-
pended in saline to a concentration of 108 CPU/ml. One-tenth milli-
liter of the bacterial suspension was added to flasks containing

10 m1 of culture medium of 90% HESS, 10% FBS and 0.0056 M glucose.
Dihydrostreptomycin was added to produce a series of graduated cen-
centrations and the flasks were incubated at 37 C in a gyrorotatory
water bath. Samples were taken at appropriate times and serially
diluted and plated on nutrient agar.

In tests involving the addition of macrophages, peritoneal
edeate was collected as described, the cells were washed twice with
sterile HBSS, resuspended in culture medium of 90% HBSS, 10% FBS and
p 0.0056 14 glucose and counted. The test suspensions were prepared as
above with the addition of enough large mononuclear cells for a final
concentration of 106 cells/ml. The final staphylococcizmacrophage
ratio was 1:1. Flasks were incubated on a gyrorotatory water bath‘. at
37 C. One milliliter samples were removed from each test flask at
appropriate times over an 8 hour period and transferred to conical
centrifuge tubes. The contents were centrifuged and 0.1 ml of the
supernatant fluid was removed for serial dilution and plating on ,
nutrient agar to determine the extracellular staphylococcal population.

Assay for bactericidal activity _o_f macronhage lysates. This

 

assay was a modification of that used by Hirsch (1958). Two days

prior to testing, the mice were injected with an immunizing dose of

20

antigen and 1.0 ml of sterile 0.1% glycogen. 0n the day of the test,
macrophages were collected, washed twice with HESS, counted and resus-
pended in 1: ml of sterile 0.25 M sucrose. Delta-hemolysin was added
to a concentration of 50 HDSO units/ml. The suspension was incubated
in an ice bath for 1 hour with a magnetic stirrer. After incubation
a small aliquot was removed and tested for completeness of lysis with
an aqueous stain of 2.1% crystal violet and 0.1% citric acid.

Solutions of lysate were diluted in 0.07 M citrate-phosphate
buffer with 0.01% ‘Bovine Serum Albumin (BSA)( Sigma Chemical Co.,
St. Louis, Mo.) to represent a lysate from 106 large mononuclear *
cells per milliliter and serial 3-fold dilutions were then made from
this solution with final test volumes of 1.0 m1. All lysate solutions
were kept on ice until the time of incubation.

An 18 hour culture of Staphylococcus aureus was centrifuged
and resuspended in saline to a calculated 2 x 103 CFO/ml. One-tenth
milliliter of the staphylococcal suspension was carefully transferred
to the lysate dilutions. The test suspensions were then incubated for
2 hours at 37 C on a 'gyrorotatory water bath. At the end of the incu-
bation period, 20 ml. of warm (’48 C), melted nutrient agar was added to
each test tube and the contents were mixed in a vortex blender. The
agar suspension was poured into petri dishes and, after solidifying,
incubated for 21; hours at 37 C before counting.

Fractionation 9_f macronhage lysate for localization 5! bacteri-

 

ggfl. activity. Two days prior to testing mice were injected with an
imunizing dose of antigen and 1 m1 of sterile 0.1% glchgen. On the
day of the test, peritoneal cells were collected, washed twice with

HESS, once with cold 0.25 M sucrose and resuspended in 5 m1 of 0.25 M

sucrose (Cohn and Wiener, 1963). Delta-hemolysin was added to a

21

concentration of 50 HDSO units/ml and the suspension was incubated in

an ice bath with stirring for 1 hour. The cell suspension was
checked for lysis as above.

One milliliter of the lysate was removed
for testing as the "total homogenate" fraction.

The remaining lysate
was subjected to an initial low speed centrifugation'(500 x'g for

12 minutes) at 0 C to obtain the "nuclear pellet".

The "nuclear
pellet" was resuspended in h m1 of citrate-phosphate buffer and 0.1% '

BSA at pH 7.0 and the supernatant fluid was centrifuged at 15,000 x‘g

for 12 minutes at 0 C to obtain the "lysosomal pellet".

The super-
natant fluid was removed and labeled "high speed supernatant" and the

"lysosomal pellet" was resuSpended in h ml of 0.071! citrate-phosphate
buffer and 0.1% BSA.

The snapensions were allowed to sit overnight

at 0 C after which the fractions were serially diluted in buffer and
the bactericidal assay repeated.

.- nu...—

-‘--ou-

RESULTS

Extracellular growth 2f staphylococci

 

 

Determination 2£.£250.2£ Staphylococcus aureus in mice via

 

 

pgritoneal route. The immunizing doses were estimated from an initial

 

LDSO test. The dose required to kill 50% of the test group was
1.3 x 107 can/m1 under the given test conditions (Table 1).

Staphylococcal growth in suSpension with peritoneal macro-

 

 

.E!E£E_° Staphylococci and macrOphages were suspended in a test

medium of 90% HESS, 10% FBS and 0.0056 M glucose in a ratio of 10:1.
No attempt was made to keep the extracellular staphylococcal population
-from multiplying. The increases in total and extracellular staphylo-
cocca1.colony forming units with time are shown in Figures 1.and 2.

No significant differences were seen and it was obvious that the

very rapid rate of extracellular growth completely masked any intra-
cellular killing which might be taking place. An attempt was then
made to control the extracellular population of staphylococci by
washing and/or the addition of antibiotic. “

Extracellular growth 2f staphylococci after repeated washing

 

 

‘gf parasitized peritoneal macrophages. In additional experiments
repeated washings of macrOphages were made in an attempt to measure the
rate of intracellular killing without the addition of antibiotic. The
results of the experiment are shown in Figure 3. In spite of the

washings, the extracellular staphylococcal population was not reduced

sufficiently to prevent interference with the measurement of

23

Table 1. The‘LDSo for Staphylococcus aureus with female Swiss

albino mice via the peritoneal route.

 

 

Calculateda Actualb Mortalityc
staphylococcal ' staphylococcal rate
population population

CPU/m1 CFU/ml
5.8 x 109 6.0 x 109 6/6
1.2 x 109 1.0 x 109 6/6
2.5 x 108 1.0 x 103 6/6
5.0 x 107 1.3 x m" 3/6
1.0 x 107 2.5 x 106 2/6

 

aPepulations of staphylococci were estimated on the basis of
optical. density versus number of colom' forming units per milliliter
measurements at 620 nm. _

bActual populations were determined by making serial dilutions
of. the suspensions of staphylococci and plating out on nutrient agar.
Plates were read at 18 hours after incubation at 37 C.

Siortality rates were based on number of animals found dead

at 20 hours compared to the total number tested. _

2h

CFU PER MI.

 

O l 2 3

HOURS

Figure 1. Total staphylococci, both extracellular and macrophage-
associated, vs. time in the presence of 106 peritoneal lar e mono-
nuclear cells/ml and without streptomycin. Control mice ( ): mice
immunized with live staphylococcus (1}): mice immunized with dead
staphylococcus (.): mice immuniz with BCG (D): staphylococcus
growth control (no macrophages) (3

25

109

CFU PER ML

 

HOURS

Figure 2. Ex racellular population of staphylococci vs. time in
the presence of 1 large mononuclear cells/ml and without streptomycin.
Control mice (0): mice immunized with live staphylococci (A): mice
immunized with dead staphylococci (.): mice immunized with BCG (Cl ):
staphylococcus growth control (no macrophages) (-).

26

Figure 3. Rate of killing of staphylococci by peritoneal
louse macrophages following repeated washings of macrophages after
incubation with staphylococci for 15 minutes at 37 C and without
streptomycin. Control mice (0): mice immunized with dead staphylo-
coccus (A): mice immunized with live staphylococcus (.): mice

imunized with 1300 (I).

27

lo8

 

10"

as mm.— :50

105
0'

HOURS

28

intracellular,and cell associated staphylococci.
Staphylococcal_g:owth lg presence pf different amounts pf

dihydrostreptomycin. The effect of different concentrations of

 

dihydrostreptomycin on the viability and growth of staphylococci is
shown in Figure b. No peritoneal cells were added in this test.
There was a very small difference in the concentrations of antibiotic
which would allow growth or would kill staphylococci. While 0.1 ug/ml
had no apparent effect, 0.5 ug/ml caused rapid killing. The effects
of different concentrations of streptomycin on staphylococci in the
presence of peritoneal macrophages is presented in Figure 5. The
growth curves represent staphylococcal concentrations in the extra-
cellular phase only. ‘With macrophages present, 5 ug/ml of dihydro-
streptomycin were required to prevent the extracellular growth of
staphylococci. This was a ten-fold increase over the concentration
required in the absence of macrophages and appeared in agreement with
the work of Bonventre and Imhoff (1970) who reported the uptake and

concentration of tritiated dihydrostreptomycin within macrophages."

Bactericidal activity pf peritoneal macrophages

 

and pgritoneal macrophage lysates

 

jIntracellular inactivation pf staphylococci By washed mouse

 

 

 

peritoneal macrophages ip suspension with 2 ug/ml dihydrostreptomycin.

 

Differencesoin the four test groups of mice were detected with the
addition of 3 ug/ml dihydrostreptomycin to a parasitized and washed
suspension. This concentration of antibiotic was chosen based on the.
above results. Using unwashed parasitized macroehages, 5.0 ug/ml
dihydrostreptomycin checked staphylococcal growth while 2.5 ug/ml did

not. In this test macronhages were washed after parasitization and a
i

CFU. PER MI.

29

 

103
0 2 4 6

HOURS

Figure h. Staphylococcal growth curve in the presence of
different amounts of streptom cin. Streptomycin concentrations:
0.05 ug/ml ( )3 0.10 tag/m1 ( )3 0.5 ug/ml (A): 1.0 ug/ml (.):
5.0 ug/ml ( .

30

CPU PER MI.

 

O 2 4 -: 6

HOURS

Figure 5. Extracellular staphylococcal growth curve in the
presence of 10 peritoneal mouse macrophages/ml and different amounts
of streptomycin. Streptomycin concentrations: 0.5 ug/ml.([]):

1.0 ug/ml (O): 2.5 ug/ml (.): 5.0 Ug/ml (v): 10.0 ug/TRI (.).

31

compromising value of 3 ug/ml dihydrostreptomycin was used in an
attempt to use as little antibiotic as possible. Viability of macro-
'phages was checked at each time period and was 95% or better throughout
the test. No significant differences in intracellular inactivation
were observed between the control mice or mice injected with either
live or dead staphylococci. There was a slightly greater rate of
bactericidal activity within macrophages of ECG immunized mice
(Figure 6).

Bactericidal activity 2; diluted machphage lysates agginst

 

staphylococci. The effect of macrophage lysates on the viability of

 

a standard population of staphylococci under various conditions are
presented in Tables 2 through 7. The dilutions marked with arrows
in these tables represent the EDSO for that lysate preparation, i.e.,
the dilution which was effective in killing 50% of the original staphylo-
coccal pepulation. No bactericidal activity was detected in macrophage
lysates from any of the four test groups in the presence of 5% mouse
serum at either pH 5 or 7 (Tables 2 and 3). It was possible that serum
proteins coated the bacterial cells and prevented their inactivation.
'Hhen 5% mouse serum was omitted, there was a definite bactericidal
effedt at pH 5 against both epsonized and unapsonized staphylococci
with macrophage lysates from all test groups (Tables h and 6). The
lysates from mice immunized with live staphylococci and B00 had a
slightly higher titer although the differences are slight. A slightly
_ higher titer was observed with unapsonized staphylococci (Table 6) as
compared to opsonized staphylococci (Table h) for all test groups. The
bactericidal activity was most likely due to lysosomal acid hydrolases
which have acid pH optima.

No bactericidal activity was detected at pH 7 against opsonized

staphylococci in lysates from control, dead, or live staphylococcal

32

Figure 6. Rate of intracellular killing of staphylococci by
peritoneal mouse macrOphages in the presence of 3 ug/ml streptomycin
following repeated washings of macrOphages after incubation with
staphylococci for 15 minutes at 37 C. Control mice (0): mice im-
munized with dead staphylococci (A): mice immunized with live

staphylococci (.): mice. immunized with BCG (I).

33

6
0
1

1... .\
5m

 

..| I;

4
0
1

as. amn— Duo

103

HOU R‘S

3h

 

 

 

Table 2. Effect of serial 3-fold dilutions of macrophage lysate on
staphylococcal viability. Test medium: 0.07 M citrate-
phosphate buffer, 0.01% 88A,: 5% mouse seruma at pH 5.
Control Dead Live ECG
mice staphylococci staphylococci immunized
immunized mice immunized mice mice
Lysate h
dilution Staphylococcal CFU
1:3 92 170 603 hon)
1:9. 102 1b6 368 536‘
1:27' 92 161 306 532
1:81 108 158 300 I 580
I 1:213 102 152 281: 608
1,729 96 15h~ 287 S90
1:2,187 103 16h 29h 63h
1:6,561 1011 11:6 286 S72
1:19,683 101 11.2 256 561;
159.0119 10h 152 301: 580
Bufferc 102 151: 320 552
control

 

aSerum used in each test group was obtained from mice immunized

in. a_ similar manner.

bNumber of staphylococcal CFU remaining at end of 2 hours incu-

bation in lysate dilutions.

cNumber of staphylococcal CFU remaining at end of 2 hours incu-

bation in buffer only.

35

Table 3. Effect of serial 3-fold dilutions of macrOphage lysate on

staphylococcal viability.

Test medium:

0.0? M citrate-

a
V phosphate buffer,— 0.01% BSA,~ 5% mouse serum at PH 7-

 

 

 

‘ .Control Dead Live ECG
mice staphylococci staphylococci immunized
immunized mice immunized mice mice
Lysate
dilutiOn Staphylococcal CFUb
1:3 220 173 521 736
1:9 173 173 h30 780
1:27 186 161 32h ‘ 76h
1:81 210 162 290 752
1:21.3 228 160 255 956
1:729 190 16h 291 768
1:2,187- 219 168 262 776
1‘:6,561 211 162 257 68!:
l:19,683 . 203 172 25k 800
1:59.0h9 226 17h 257 7&8
Bufferc 180 168 261 765

control

- .—

‘Serum used in each test group was obtained from mice immunized

in a similar manner.

bNumber of staphylocOCcal CFU remaining at end

bation in lysate dilutions.

of 2 hours incu-

-.

cNumber of staphylococcal CFU remaining at end of 2 hours incu-

bation in buffer only.

36

Table h. Effect of serial 3-fold dilutions of macrophage lysate on
viability of opsonized staphylococci.a Test medium: 0.07 M

citrate-phosphate buffer and . 0; 01%TBSA'at‘ 'pH . 5.

 

Control Dead Live T ‘ ECG

 

 

mice ' staphylococci staphylococci immunized
immunized mice immunized mice mice
21312:... 2 Staphylococcal C‘F'Ub
d . »- . .- .4
1:3 69 __ 220 {'83 . 101:
1:9 _ 100 233 11:6 ’ 27
1:27 83 1% .- 75 3
1:81, 101 >187 37 ‘ . >.78
1:2113 ' 12!: 21:5 >311 318
1:729 ._ ' 116 260 200 1.31.
1:2,187 115 258 152 1a.
1:6,561 ’ 122 252 200 1:12
1:19,683 125 275 2511 1:33
1:59.019 ‘ 113 263 198 ' 36h
Bufferc 117 ' 256 217 109

control

 

IIStaphylococci were opsonized with mouse serum from a". cane-
torresponding test group.
’ bNumber of staphylococcal CFU remaining at end of 2 hours incu-
bation in lysate dilutions.
clumber of staphylococcal CFU remaining at end of 2 hours incu-
bation in buffer only.

63050, lysate dilution which kills 50% of added staphylococci.

37

Tdble 5. Effect of serial 3-fold dilutions of macrophage lysate on
viability of opsonized staphylococci.b Test medium:

0.07 M citrate-phosphage buffer and 0.01% BSA at pH 7.

 

 

 

Control Dead Live ECG
mice staphylococci staphylococci immunized-
immunized mice immunized mice mice

hysate b
dilution Staphylococcal CFU
1:3 - ’ 139 316 553 361
1:9 112 261 301 11:9
1:27 105 227 261: 131
1:81. . , 100 - 272 l 2 281: 168
1:21.3 110 258 296 ‘ .263
. 1:729 4 115 210 280 322
1:2,187 . 110 I 21.0 296 339
1:6j‘561 - 105 21.2 318 390 .
1:19,683 ‘ 1111 261 311: 368
1:59.0119 130 205 286 33!:
Bufferc . 108 263 ‘ 326 335
control

 

,TStaphylococci were 0psonized with mouse serum from a corres-
ponding test group.

bNumber of staphylococcal CFU remaining at end of 2 hours incu-'
bation in lysate dilutions.

cNumberor staphylococcal CFU remaining at end of 2 hours incu-
bation in buffer only.

dEDSO, lysate dilution which kills 50% of added staphylococci in

2 hours.

38

Table 6. Effect of serial 3-fold dilutions of macrophage lysate on
viability of staphylococci. Test medium: 0.07'H citrate-

phosphate buffer and-0.01% BSA at pH.5.

 

 

 

Control Dead Live ECG
mice ' staphylococci staphylococci immunized
immunized mice .immunized mice mice
lysate
dilution 1 Staphylococcal CFUa
1:3 27 158 0 1
1:9 ' 7 175 119 o
1:27 , 27 91 79 0
1:81 c > 39 89 15 2
1:213 105 11:1: 11 ' 131:
, > >

1:729 ‘ 130 2211 > 63 28h
1:2,187 138 259 112 h77
1:6 7,561 13!: 237 166 510
1219,683 129 205 1b0 378
1:59.019 132 258 137 11118
Bufferb 121: 237 1215 1:51:
control

 

‘Number of staphylococcal CFU remaining at end of 2 hours incu-
bation in lysate dilutions..

bNumber of staphylococcal CFU remaining at end of 2 hours incu-
bation in buffer only.

cEDSO’ lysate dilution which kills 50% of added staphylococci.

in 2 hours.

39

Table 7. Effect of serial 3-fold dilutions of macrophage lysate on
staphylococcal viability. Test medium: 0.07 M citrate-g

phosphats buffer and.0;01%‘ESA at pH.7.

‘ r
w...
~ .
s ‘ ,..‘ g
‘ an ,_

 

Control . Dead Live BOG
mice staphylococci staphylococci immunized
immunized mice 'immunized mice mice

 

 

hysate
dilution -. \ Staphylococcal CFUa
1:3 172 186 . 366 1:95
1:9 129 182 30!: 369
1:27 . 119 211 222 o
’1:81 123 156 278 c:> 6
1:213 121 200 p 232' ~ 355
1:729 ‘ 118 132 272 313
1:2,187 122 121‘: 2116 392
1:6;561 119 206 230 1:16
1:19,683 88 192 171: 1:09 ,
1: 59,0119 129 189 252 1:25
Bufferb 115 _ 179 255 1:11
control ' '

 

‘thber of staphylococcal CFU remaining at end of 2 hours incu-
bation in lysate dilutions.

”Number of staphylococcal CFU remaining at end of 2 hours incu-
bation in buffer only. -

cEDSO, lysate dilution which kills 50% of added staphylococci.

in 2 hours.

ho

imunized mice although there was a slight effect with macrophage
lysates from ECG immunized mice (Table 5). There was definite
bactericidal activity against opsonized staphylococci at pH 7 by
lysates from ECG immunized macr0phages, the positive control group
(Table 7).

All tests in this series were made in triplicate and the
figures represent averages of these values. Buffer controls were
made with each test series and represent the number of staphylococci
remaining viable in buffer alone. Although staphylococci were added
' to a calculated 200 CFU/ml in each test system, plate counts indicated
some deviation. Dysate controls were also run with each series. One
milliliter of lysate was added to the appropriate buffer and treated
in the same manner as the test systems. Except for macrophages
from mice injected with live staphylococci, no contamination was
found associated with the cell lysate. 'Mice from this test group had
been prestimulated with 107 live staphylococci two days prior to
testing and some viable staphylococci were invariably found in the
lysate contents. The values found in the tables representing this
test group have been normalized by subtracting a calculated correction
factor, i.e., one-third of the lysate control staphylococcal plate
count was subtracted from the plate count obtained for the 1:3 dilution,
one-ninth of the lysate control staphylococcal plate count was sub-
tracted from the plate count obtained for the 1:9 dilution, etc.

Localization pf bactericidal activity 12 macrophage lysates

 

 

 

 

from BOG immunized mice. The presence of an active factor(s) at
pH 7 from lysates of ECG immunized mice prompted an experiment to

determine a possible association with lysosomes. Lysate from this

81

test group was separated in0.25 M sucrose by differential centri-
fugation into three fractions: (1) a "nuclear" fraction, (ii) a
“lysosomal" fraction, and (iii) the remaining "high speed super-
natant” (Cohn and Wiener, 1963). Serial 3-fold dilutions were made
with these fractions and part of the total homogenate and their
bactericidal activities tested against a standard concentration of
staphylococci. It appeared the major portion of the bactericidal
activity was in the lysosomal fraction although a significant killing
effect was also found in the supernatant fluid left from the high
speed centrifugation (Table 8). It was possible the lysosomal mem-
branes ruptured during treatment and released their contents into the
nonsedimentable fraction.

c"§££gg§‘2£ staphylococcal challenge pp survival 2: immunized
.2322. The four groups of mice were challenged with 2 x 109 CFU
(100 L050) of staphylococci via the peritoneal route three days after
immunization. The test group immunized with live staphylococci
survived challenge best: 33% were dead by 20 hours (Table 9). The
ECG and dead staphylococci immunized group had high death rates,
93% and 83% respectively. Control mice had a 100% mortality rate.

L2

Table 8. Effect on staphylococcal viability of serial 3-fold dilu-
tions of fractions from macr0phage lysate obtained by
differential centrifugation. Source of macr0phages: ECG
inununized mice. Medium: 0.07 H citrate-phosphate buffer and

 

 

Fraction
Lysate‘ V Total Nuclear Lysosomal High speed
dilution homogenate pellet pellet supernatant

 

' Staphylococcal CFUa

 

1:3 329 167 1:11 ' 211
1:9 183 127 1: _. _ 110 I
1:27 7 158 :6 . 2h h
1:81 69 0 . 71 1
1:2h3 29 57 0 26
1:729 0 180 0 30
1:2,187 3 126 0 27 _
1:6,561 11 151. o 17
1:19,683 116 150 0 o
1:59.089 12 130 57 ' I ’ 3~

 

‘Number of staphylococcal CFU remaining at end of 2 hours incu-

bation 1“ lysate dilutions. The number of staphylococci remaining at

the end of 2 hours incubation in buffer only was 210 CFU.

h3

Table 9. Effect of a challenge dose of staphylococci on the survival

of test mice.

 

 

Mice No. of mice dead/ a Survival

test No. of mice tested rate
groups '
Control 18/18 0%
Dead - 13/1h 7%
staphylococci

immunized

Live 6/18 ' 67%
staphylococci

immunized
ECG 15/18 . 17%
immunized

 

a
Mortality rates are on the basis of number of animals dead

at 20 hours.

DISCUSSION

Cellular immunity and the problem 2: antibiotics. A cellular

 

 

immunity has been demonstrated for several kinds of microorganisms
including mycobacterium, salmonella, brucella, listeria, toxoplasma,
and 1eishmania.(Mackaness, 1968: Dubos and Schaedler, 1957: Coppel

and Youmans, l969A,vl969E: Youmans and Youmans, 1969: Jenkin and
Rowley, 1963; Miller and Twohy, 1969). In all cases the agent inducing
a cellular immunity was an absolute or facultative intracellular
parasite and with the exception of mycobacterium immunity was induced
only with living organisms.

. Instances of survival or degradation only over an extended
period of time was demonstrated for staphylococci within monocytes
(Shayegani and Kapral, 1962) and intracellular survival and multi-
plication was observed for a relatively nonvirulent strain of Serratia

marcescens Giiller and Buckler, 1968) although a cellular response

 

has not been demonstrated for either organism.

Our findings (Fig. 3) concurred with those of Kapral and
Shayegani (1°59) in which it was demonstrated that washing the host
cells after parasitization was rather ineffective in controlling
the extracellular growth of staphylococci for the purpose of
studying intracellular inactivation of staphylococci. These high

‘

concentrations of viable extracellular Staphylococcup aureus

 

hh

hS

prevented any determination of differences in intracellular killing
between normal and immunized animals. 'When 3 ug/ml of antibiotic

were incorporated into a test system measuring intracellular degra-
dation of washed macr0phages, an increased rate of inactivation was
observed in ECG immunized mice (Figure 6). Little difference in
staphylococcal inactivation was observed between normal mice or'mice
immunized with dead or live staphylococci. Approximately a 33% decrease
in colony forming units was observed after zero time between the
positive controls (EGG immunized mice) and the other test groups.
After an initial one hour period of intracellular degradation approxi-
' mately 1.5 x 108 CFU of staphylococci were associated with lOé large
mononuclear cells from ECG immunized mice, while about 5 x 10b CFU 5‘
-were found with the-same_number of cells in the-other test groups.
Feasibly the macrophages from ECG immunized mice took up dihydro-
streptomycin to a greater degree than macrophages from other groups
and this additional amount might account for the differences observed
in this test: however, the possibility of a real effect must also be
considered.

Bactericidal activity pf immune versus normal macrophgges.

 

Much work was been published about the rate of intracellular survival
or inactivation of invading organisms by cells of the reticuloendo-
thelial system (Lurie, 1939, Li et al., 1963: Mackaness, l96h: Rowley,
1958) and the hydrolytic action of lysosomal enzymes has been well
documented (Cohn, 1963 A, 1963 B: Friedberg and Shilo, 1970: Friedberg
et al., 1970). Degradation of ingested proteins, macromolecular
carbohydrates and bacteria by lysosomal enzymes has been established
(Aronson and DeDuve, 1968: Coffey and DeDuve, 1968: Ehrenreich and

' Cohn, 1969). Shayegani (1968) noted a bactericidal action of the

a 0 .

D6

lysosomal contents from polymorphonuclear leucocytes on staphylococci.

Animals which have been immunized with 800 have elevated levels
of lysosomal acid hydrolases (Cohn and Wiener, 1963: Saito and Suter,
1965) which might explain their elevated bactericidal activities in
cellular immunity. Other investigators (Dannenberg and Bennett, 1963)
have found no increase in levels of enzymes.

The experiments performed with serial dilutions of macrophage
lysates showed bactericidal activity against both untreated and
apsonized staphylococci in all test groups at pH 5 (Tables h and 6).
The E050 was at a lower titer in the case of cpsonized staphylococci.
This was in disagreement with the findings of Donaldson et al. (1956)
and Rowley (1958) in which immune serum enhanced the killing of

Escherichia coli within phagocytic cells. Cohn (1963 B) found, however,

 

that specific antibody inhibited the degradation 0f.§°.22}$° In our
experiments there appeared to be inactivation of both opsonized and
unopsonized staphylococci at greater lysate dilutions with macrophages
from live staphylococci immunized mice than with the other test groups,
but the difference between groups was not great (Tables h and 6). The
fact that killing was observed at pH 5 and not at pH 7 would seem to
implicate the lysosomal acid hydrolases which have their pH optima in
this region (Aronson and DeDuve, 1968; Coffey and DeDuve, 1968). In
experiments with alveolar macrOphages, Cohn and Wiener (1963) found
similar increases in lysosomal enzyme concentrations.’ In normal
alveolar'macrOphages unit activity per 10'6 macrophages for acid phos-
phatase was 20.7 ug phosphorus/hr at 38 C, for lysozyme it was 3.2 ug
egg white lysozyme equivalents at 22 C, and for-lipase it was 0.31 "~
umoles napthol/hr at 38 C. In 800 induced alveolar macrOphages the

corresponding activities were 37.0 for acid phosphatase, 9.2 for.

b7

lysozyme and p.83 for lipase.

A.bactericidal factor(s), presumably lysosomal, obtained from
peritoneal cells of ECG immunized mice was active against staphylo-
cocci at pH 7 (Table 7). This factor(s) may or may not have been
produced by the macrophages. Patterson and Youmans (1970) have observed
the presence of a factor(s) apparently produced by immune lymphocytes
and taken up by macrOphages. The macrophages then exhibited an
increased bactericidal activity. In our tests, (Table 7) no bacteri-
cidal activity was observed at pH 7 in any of the other test groups.

In fractionation of the macrOphage lysate, most of the bactericidal
activity was located in the "lysosomal pellet" although some activity

m was observed in the "high speed supernatant". This latter activity
'night have been due to the inadvertant rupturing of lysosomal.membranes
during handling and treahment with the resultant "spilling” of the
lysosomal contents. Further tests should be made to characterize this
substance(s). .

This bactericidal effect at pH 7 might account for the difference
in intracellular killing within intact macrophages of BOG immunized

'mice as compared with the other test groups.

A

SUMMARY

A slightly greater intracellular killing was observed in
macrophages from BCG immunized mice (positive controls) than in
macrophages from control and dead and live staphylococcus immunized
nice. A small amount of dihydrostreptomycin was incorporated into
the test medium, however, and the observed differences may be due to
a greater uptake of antibiotic by the immune macrophages from the
positive control group.

Killing of opsonized and unopsonized staphylococci in the
presence of macrophage lysates was observed in all test groups at

pH 5 with a slightly higher ED obtained for animals immunized with

50
live staphylococcus or BCG. ‘Hice immunized with BCG produced (or
exhibited) a factor(s) which was probably lysosomal in origin and active
at pH 7.in.bactericidal activity against staphylococci. This bac-
tericidal effect was not seen in control.mice or mice immunized with

either live or dead staphylococci at this pH.

he

in! IA] i‘llllliil '1'"
7" III! I.

ADDENDUM

1. It was decided after all experiments were finished that
an extra test group might well have been included here. A group of
test mice which received one staphylococcal injection prior to
testing, similar to the immunization schedule of the mice immunized
with BOP; might have been added.

2. although Swiss albino female mice were specified for use

as test animals, the original mice with which the LDSO dose of.

' ‘1‘

staphylococci was determined and the actual test mice were supplied

‘ by different breeders. It was subsequently discovered that mice

obtained from Carworth Farms (those with which the LDSO was determined)

provided a more uniform and predictable response than those obtained
from Spartan Research (those with which the body of this research was
performed). I

h9

[llit I‘ll-Ill!!! ll-"Illlllll'llll

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