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Isolation and characterization of group A streptococcal Fc receptors

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Isolation and characterization of group A streptococcal Fc receptors
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Yarnall, Michele S., 1959-
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ix, 91 leaves : ; 29 cm.

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Antibodies ( jstor )
Bacteria ( jstor )
Cellulose nitrate ( jstor )
Fc receptors ( jstor )
Gels ( jstor )
Humans ( jstor )
Immunoglobulins ( jstor )
Reactivity ( jstor )
Receptors ( jstor )
Staphylococcus aureus ( jstor )
Receptors, Fc -- isolation & purification ( mesh )
Streptococcaceae ( mesh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
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Includes bibliographical references (leaves 82-90).
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Typescript.
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Vita.
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by Michele S. Yarnall.

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ISOLATION AND CHARACTERIZATION OF GROUP A
STREPTOCOCCAL Fc RECEPTORS







By

MICHELE S. YARNALL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1985




ISOLATION AND CHARACTERIZATION OF GROUP A
STREPTOCOCCAL Fc RECEPTORS
By
MICHELE S. YARNALL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGkEE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1985


ACKNOWLEDGEMENTS
I wish to express my thanks to Dr. Michael D.P. Boyle for giving
me the chance to work in his laboratory. I appreciate all of his help
and guidance and have enjoyed working with him.
I would like to thank my committee members, Dr. E.M. Ayoub, Dr.
A.P. Gee, Dr. J.W. Shands, Dr. A.S. Bleiweis, Dr. J.B. Flanegan, and my
external examiner, Dr. A.D. O'Brien, for their helpful suggestions
throughout this study.
I would also like to thank Dr. Frank Muzopappa for his
encouragement and advice throughout rny years at Kutztown State
Col lege.
I wish to give my appreciation to my family who has been
constantly supportive of me. I would especially like to thank Kim to
whom I can always talk and who has been great company on many
vacations.
Finally, I would like to thank David C. Palmer. His love,
patience, and understanding has helped me through some difficult
ti mes.
i i


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT viii
CHAPTER
ONE INTRODUCTION 1
Staphylococcal Protein A The Type I Fc Receptor 1
Streptococcal Fc Receptors Type II Through Type V 4
Fc Receptors as Virulence Factors 7
Practical Applications of Bacterial Fc Receptors 8
Summary 9
TWO A TECHNIQUE FOR THE DETECTION OF BOUND AND SECRETED Fc
RECEPTORS 11
Introduction 11
Materials and Methods 12
Results 14
Discussion 24
THREE ISOLATION AND PARTIAL CHARACTERIZATION OF THE TYPE II Fc
RECEPTORS FROM A GROUP A STREPTOCOCCUS 27
Introduction 27
Materials and Methods 28
Results 36
Discussion 60
FOUR DISTRIBUTION OF THE TYPE II Fc RECEPTORS ON NEPHRITOGENIC
AND NN-NEPHRITOGENIC GROUP A STREPTOCOCCI 65
Introduction 65
Materials and Methods 66
Results 67
Discussion 72


Page
FIVE CONCLUSION 79
REFERENCES 82
BIOGRAPHICAL SKETCH 91


LIST OF TABLES
TABLE Page
1-1. Species and Subclass IgG Reactivities of Bacterial
Fc Receptors 5
3-1. Fc Receptor Activity in Streptococcal Extracts 38
4-1. Binding of 125i Human IgG to Nephritogenic and
Non-Nephritogenic Group A Streptococci 70
4-2. Interaction of Nephritogenic and Non-Nephritogenic
Group A Streptococci with Hunan IgG Subclasses 73
v


LIST OF FIGURES
FIGURE Page
2-1. Schematic representation of the immunoblotting
procedure for detection of surface Fc receptors 16
2-2. Detection of Fc receptors on the surface of
staphylococcal strains 17
2-3. Sensitivity of assay for secreted Fc receptors 19
2-4. Fc receptor expression of a group A streptococcal
strain 20
2-5. Fc receptor expression of two bacterial subpopulations
selected from the original strain 64/14 22
2-6. Fc dependent uptake of 125j_-|abeiecj human IgG by
increasing numbers of bacteria 23
3-1. Western blot autoradiograph of affinity purified
extractions of 64/14/HRP 39
3-2. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of the type II Fc receptor 41
3-3. The effect of dipeptides on the binding of
125i_iabeled human IgG subclasses to an Fc
receptor-positive group A streptococcus 43
3-4. The effect of glycyl-histidine on the binding of
125l-labeled human IgG to three bacterial strains 44
3-5. Human immunoglobulin G subclass reactivity of
bacterial Fc receptors 46
3-6. Reactivity of three human myeloma IgG3 samples with
bacterial Fc receptors 47
3-7. Inhibition of binding 125j_-|abe1 ed 1963 to a
group A streptococcus by unlabeled human IgG
subclasses 49
3-8. Separation of the type I la Fc receptor from the type
11b Fc receptor 51
VI


FIGURE Page
3-9. Inhibition of 125j human IgG or 125¡ human
IgG3 to group A strain 64/14/HRP by immunogloubl in G
from a variety of mai.malian species 53
3-10. Reactivity of IgG from a variety of species with the
type I la or type 11 b Fc receptor 55
3-11. Inhibition of binding of 125j-labeled human IgG
or 125i-labeled human IgG3 to 64/14/HRP by
monospecific antibodies against the purified type I la
or type lib Fc receptors 58
3-12. Inhibition of binding of 125i human IgG to
64/14/HRP by antibody against the type I receptor,
the type II receptor, or the type III receptor 59
4-1. Binding of 125i human IgG to nephritogenic and
non-nephritogenic group A streptococci 69
4-2. Proposed mechanism of the pathogenesis of
post-streptococcal glomerulonephritis 75
vi i


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ISOLATION AND CHARACTERIZATION OF GROUP A
STREPTOCOCCAL Fc RECEPTORS
By
Michele S. Yamal 1
August 1985
Chairman: Michael D.P. Boyle, Ph.D.
Major Department: Immunology and Medical Microbiology
An immunoblotting technique v/as developed to select a group A
streptococcal strain rich in Fc receptors. A high Fc receptor-positive
strain was selected and used as the source for the isolation of two
functionally active Fc receptors. A variety of extraction techniques
were compared including 1) heat extraction at neutral, acid or alkaline
pH, 2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin,
papain or phage lysin, or 3) autoclaving or heating in the presence of
sodium dodecyl sulfate. The most homogeneous receptor was recovered
following heat extraction and contained two molecular weight forms.
One form had a molecular weight of 56,000 dal tons and the other form
had a molecular weight of 38,000 daltons. The 56,000 dalton Fc
receptor was capable of reacting with human IgG subclasses 1, 2, and 4,
pig IgG and rabbit IgG. The 38,000 dalton Fc receptor could only bind
human IgG subclass 3. The two Fc receptors could be separated by
binding to and elution from a column of immobilized human IgG3 which
resulted in the isolation of the 38,000 dalton Fc receptor. The


unbound material from the immobilized IgG3 column was applied to an
imnobilized column of human IyG and the 56,000 dal ton Fc receptor was
recovered following elution. Both Fc receptors were antigenical ly
related. Monospecific antibodies prepared against either the 56,000
dal ton Fc receptor or the 38,000 dal ton Fc receptor demonstrated
reactivity with both molecular weight forms. Both group A
streptococcal Fc receptors were found to be antigenically and
physicochemically distinct from either the type I receptor found on the
majority of Staphylococcus aureus strains or the type III Fc receptors
found on the majority of yroup C streptococcal strains.
The distribution of Fc receptors on group A streptococci recovered
from patients who developed post-streptococcal glomerulonephritis was
tested. A high incidence of Fc receptor positive nephritogenic strains
were found, but an absolute correlation between Fc receptor expression
and pathogenic potential could not be established.


CHAPTER ONE
INTRODUCTION
Certain bacteria are capable of interacting with immunoglobulins
in two distinct ways. One involves a specific antigen-antibody
reaction in which the F(ab) portion of the immunoglobulin molecule
participates. This interaction mediates the clearance of bacteria from
the host. The second interaction involves the Fc portion of the
immunoglobulin molecule. It has been reported that certain
staphylococci (Jensen, 1953; Forsgren and Sjoquist, 1966) and
streptococci (Kronvall, 1973a) have surface receptors that are capable
of reacting with the Fc region of certain classes and subclasses of
immunoglobulins. Functional studies of their reactivity with different
species and subclasses of IgG have suggested that five distinct
bacterial Fc receptor activities exist (Myhre and Kronvall, 1981).
These are designated as types I through V.
Staphylococcal Protein A The Type I Fc Receptor
Physicochemical Properties
The most extensively studied Fc receptor is the type I receptor
isolated from Staphylococcus aureus and designated protein A. Protein
A has been reported to be produced and or secreted by approximately 90%
of all staphylococci studied (Langone, 1982a; Sperber, 1976); however,
marked quantitative differences exist between different strains. The
most widely studied Staphylococcus aureus Cowan I strain is rich in
surface protein A whereas the Staphylococcus aureus Wood 46 strain
1


2
expresses very low levels (Freimer et_al_., 1979; Reis et_ aU, 1984a).
Isolation of protein A by lysostaphin extraction results in a
homogeneous product which is composed of a single polypeptide chain
with a molecular weight of 42,000 daltons (Sjoqui st et al_., 1972, Bjork
et a]_., 1972). The Fc binding part of the protein consists of four
repetitive subunits. Recently, the gene for protein A has been
identified and cloned (Uhlen et_ al_., 1984; Duggleby and Jones, 1983;
Lofdahl et al_., 1983). The DNA sequence has revealed a fifth region,
homologous to the four repetitive subunits. This region, however, does
not appear to bind the Fc region of immunoglobulins. Protein A has an
elongated structure which results in anomalous estimates of molecular
weight by gel filtration (Bjork et al_., 1972 ). It has been purified in
high yields from bacterial culture supernatants, and extracts of
staphylococci, by affinity chromatography, using columns of immobilized
rabbit or human IgG (Kronvall, 1973b).
Biological Properties
The binding of protein A to the Fc region of IgG mediates a
variety of biological activities. Complexes between protein A and IgG
are capable of activating the classical complement pathway (Stahlenheim
et aj_., 1973). Addition of protein A to human (Kronvall and Gewurz,
1970; Stahlenheim et _al_., 1973), guinea pig (Sjoquist and Stahlenheim,
1969), rabbit (Stahl enheim et_ al_., 1973), dog or pig serum (Kronvall
and Gewurz, 1970) was shown to deplete complement activity. This
depletion depended on the amount of protein A relative to IgG. Langone
et _al_. (1978a,1978b) demonstrated that protein A and IgG formed
complexes that behaved functionally like IgM in their interaction with
both whole complement and purified Cl. The molecular formula of these


3
IgM-like conplexes is [(IgG)2PA32 (Langone et _al_., 1978b; Hanson
and Schumaker, 1984). Several studies have shown that formation of
these complexes between protein A and IgG inhibited phagocytosis of
Staphylococcus aureus, by suppressing the opsonizing ability of
complement (Dossett et al_., 1969; Forsgren and Quie, 1974; Peterson et
al., 1977; Verhoef et^ al_., 1977; Musher et_ al_., 1981).
Protein A is also capable of stimulating T- and B-cell
mitogenesis. The nature of this response, however, depends on whether
protein A is added in a soluble or insoluble form. Several studies
have shown that soluble protein A can act as a potent T-cell mitogen,
but can only activate B-cells in the presence of helper T-cells
(Gugliemi and Preud'Honrne, 1980; Schuurman et_ al_., 1980; Dosch et al.,
1980). Smith et jfL (1983) suggested that the T-cell mitogenic
activity was not associated with protein A, but was due to a bacterial
exotoxin.
In contrast to soluble protein A, protein A on intact bacteria, or
bound covalently to sepharose or sephadex particles, is primarily a
T-cel 1 -independent, B-cell mitogen (Romagnani _et aK, 1980; Ruuskanen
et_ aj_., 1980; Pryjma et_ al_, 1980a,b). Insoluble protein A can
stimulate the secretion of IgM, IgG, and IgA by both peripheral blood
lymphocytes (Muraguchi et^al_., 1980; Romagnani et_al_., 1980; Gausset et
al., 1980), and spleen cells (Ri ngden et al_., 1977).
In vivo studies have shown that protein A mediates immune
reactions in a manner similar to that of antigen-antibody conplexes.
Some of these effects appear to involve complement activated by the
protein A-IgG complexes (lawman et _al_., 1984). Anaphylaxis-like
reactions and Arthus reactions are seen when as little as 10 ug of
protein A is injected intradermally in guinea pigs (Gustafson et al.,


4
1968). Larger amounts of protein A (50U-100 yg) cause fatal
anaphylactic shock. In humans, an intradermal injection of 10 yg of
protein A causes wheal and erythema reactions (Martin et_ a]_., 1967).
Streptococcal Fc Receptors Type II Through Type V
Certain strains of group A, C, and G streptococci have been
reported to have Fc receptors analogous to protein A (Kronvall,
1973a). This was first recognized by the ability of these organisms to
agglutinate red blood cells sensitized with a sub-agglutinating dose of
antibody (EA), indicating the presence of an IgG Fc receptor on the
surface of these bacteria (Kronvall, 1973a). This Fc reactivity was
further confirmed by measuring non antigen-specific binding of
radiolabeled human myeloma IgG to these bacteria. Based on the ability
of sera from different animal species to inhibit the binding of
radiolabeled human IgG to a variety of bacteria, several distinct types
of streptococcal Fc receptors (FcR) have been identified (Myhre and
Kronvall, 1977). The receptor characteristic of group A streptococci
is the FcR type II. Groups C and G streptococci carry a common, or
related receptor, designated type III. Class IV FcR is found only on
bovine group G s-hemolytic streptococci, and type V is found on certain
strains of Streptococcus zooepidemicus (Myhre and Kronvall, 1981). See
Table 1-1.
Only one of the four types of streptococcal Fc receptors has been
purified and characterized. This is the type III receptor found on
certain group C and yroup G streptococci. Heat treatment (Freiner et
al., 1979), hot acid extraction (Havlicek, 1978; Muller and Blobel,
1983), phage lysis (Reis et jil_., 1984a; C hr i sterisen and Holm, 1976),
papain (Bjorck and Kronvall, 1984), trypsin or mutanolysin digestion


5
TABLE 1-1
Species and Subclass IgG Reactivities of Bacterial Fc Receptors3
Immunoglobulin
I
IgG Fc Recepto
II III
r Typeb
IV
V
Human
igGi
+++
+++
+++
+
+++
IgG2
+++
+++
+++
-
+++
IgG3
-
+++
+++
-
-
IgG4
+++
+++
+++
+
+++
Mouse
igGi
+

+
NTC
NT
IyG2a
+++
-
+++
NT
NT
I(jG2b
+++
-
+++
NT
NT
igc3
+++

+++
NT
NT
Cow
igGi
-
-
+++
-
-
IgG2
+++

+++
+
Sheep
igGi
-
-
+++
-
-
igG2
+++
-
+++
-
+
Goat
IgGi
+
-
+++
-
+
igG2
+++

+++

+
Horse
IgG(ab)
+
-
+++
-
+
igG(c)
+
-
+++
+
+
igG(T)
-
(+)
(+)
(+)
(+)
Rabbit
IgG
+++
+++
+++
NT
+++
Guinea Pig
IgG
+++
-
+++
NT
NT
Rat
IgG
-
-
-
NT
-
Dog
IgG
+++
-
-
NT
-
Cat
IgG
+++
-
-
NT
-
Pig
IgG
+++
+++
+++
NT
+++
Chicken
IgG
-
-
-
NT
NT
+++ = Indicates strong reactivity
+ = Indicates low reactivity
(+) = Weak, atypical reactivity
3 = Summarized from Kronvall, 1973a; Myhre and Kronvall, 1977,
1980a,1981; Reis, 1984c.
b = See text
c = NT, Not tested


6
(Reis et a]_., 1985) have been used to isolate this Fc receptor.
Digestions with papain or trypsin yield a homogeneous product with
molecular weights of 29 K with papain, or 40 K with trypsin.
Considerable heterogeneity in size of the type III Fc receptor, ranging
from 30,000 to 100,000 dal tons, is observed with the other extraction
procedures; however, all molecular weight species are antigenically
related (Reis et al_., 1984a), indicating that they are oligmers or that
cell wall components are attached. The streptococcal FcR type III has
a very broad reactivity, including all four subclasses of human IgG and
many other mammalian classes and subclasses (Myhre and Kronvall, 1980b;
Myhre and Kronvall, 1981; Reis et j3l_., 1984b; Boyle, 1984). The
biological properties of the type III receptors have not been
extensively characterized.
Little information is available on the type II FcR found on
certain group A streptococci. Attempts to isolate this receptor have
met with limited success for several reasons. Less than 50% of the
group A streptococcal strains tested have detectable Fc receptors
(Kronvall, 1973a; Freimer et aj_., 1979), whereas, the type III Fc
receptor is found on greater than 80% of the group C and group G
streptococcal strains tested (Myrhe and Kronvall, 1979; Myrhe and
Kronvall, 1980b). The type I Fc receptor is found on approximately 90%
of all Staphylococcus aureus strains tested (Langone, 1982a; Sperber,
1976). The amount of Fc receptor on the surface of positive strains of
group A streptococci is much less than that found of the surface of
Staphylococcus aureus or group C streptococci (Christensen and Oxelius,
1974; Freimer et aj_., 1979; Reis et 1983). In addition to low
amounts of Fc receptors on group A streptococci, these Fc receptors


7
have been reported to be unstable duriny subculture (Freimer et al.,
1979). Havlicek (1978) has reported that Fc receptors are found more
frequently on fresh isolates than on freeze-dried strains. Only 30% of
the freeze-dried strains had Fc receptors, while all of the fresh
isolates tested in this study had detectable Fc receptor activity.
The type II Fc receptor, unlike protein A or the type III Fc
receptor, is restricted in its reactivity with mammalian immuno
globulins, reacting only with the four subclasses of human IgG, rabbit
IgG, and pig IgG (Myhre and Kronvall, 1977;1980a,b) (see Table 1-1).
It has recently been reported that, based on morphological evidence,
different types of IgG-Fc receptors exist on group A streptococci
(Wagner et al_., 1983). Wagner's electron microscopy studies show that
ferritin-conjugated IgG from various species result in different
labeling patterns. Studies on the inhibition of binding of homologous
versus heterologous IgGs are also in agreement with the existence of
more than one type of IgG Fc receptor on the same strain.
The group A streptococcal IgG Fc receptors are distinct from
several cell wall constituents, including the M-protein, group carbo
hydrate, peptidoylycan (Christensen et_ al_., 1979), and lipoteichoic
acid (Schal en et d]_., 1980). In addition to IgG Fc receptors, some
strains of group A streptococci are capable of binding the Fc region of
human IgA through a distinct, receptor (Christensen and Oxelius, 1975;
Kronvall et_ al_., 1979; Schal en et_ a]_., 1980).
Fc Receptors as Virulence Factors
The involvement of Fc receptors in the pathogenicity of disease is
unknown, although some evidence suggests that there is a correlation
between the virulence of certain group A streptococci and their ability
to bind the Fc portion of human IgG (Burova et jal_., 1980,1981). Serial


8
mouse passages of various group A strains have been reported to result
in the selection of highly virulent variants. A majority of these
strains have enhanced expression of certain surface proteins, including
IgG Fc receptors, indicating the possibility of an association between
Fc receptor activity and virulence (Burova et jil_., 1980,1981). Recent
evidence indicates that group A streptococcal FcR activity can be
induced, or its expression enhanced, in association with other factors
that are known to relate to virulence. The role of plasmids in the
expression of anti-phagocytic activity, opacity factor, and IgG and IgA
Fc receptors lias been explored (Burova et_ aj_., 1983). Burova has found
that these factors may be triggered by insertion of plasmid DMA into
the bacterial chromosome. Plasmid control of the group A Fc receptors
offers an explanation of why these receptors are lost during subcultur-
iny. Before the importance of Fc receptors as virulence factors can be
critically assessed, however, this receptor will have to be isolated,
and methods developed to quantify them on fresh isolates and within
immune complexes.
Practical Applications of Bacterial Fc Receptors
The ability of certain bacterial surface proteins to react
selectively with the Fc portion of immunoglobulin molecules has been
utilized in a variety of immunological procedures primarily for
purifying and quantifying reactive classes and subclasses of IgG
(Lanyone, 1982b; Boyle, 1984; Godiny, 1978). These receptors can be
labeled to high specific activity or immobilized v/ithout loss of Fc
binding activity and can be used to detect and quantify antigens,
antibodies, or antigen-antibody complexes (Langone et _al_., 1979; Gee
and Lanyone, 1981). Currently, by using type I and type III bacterial


9
Fc receptors, virtually any antibody of the IgG class, with the excep
tion of those raised in birds or rats, can be detected. This enables a
single tracer to be used in many assays for different antigens. In
addition, binding of an electron derise ligand to Fc receptors can be
used in a variety of techniques for locating and quantifying antigen-
antibody complexes (Goding, 1978; Gee and Langone, 1981). Isolation of
the type II, type IV, and type V bacterial Fc receptors with restricted
reactivities would enable techniques to be developed that can focus on
a narrower range of immunoglobulin species, classes and subclasses.
Summary
The binding of immunoglobulins via the Fc region to certain
bacteria has been known for several years. Several studies involving
the binding of various species1 classes and subclasses of immuno
globulins to staphylococci and streptococci have determined that at
least five distinct bacterial Fc receptors exist. Only two of these
bacteria Fc receptors, however, have been purified to homogeneity and
characterized. These receptors are the type I Fc receptor from
Staphylococcus aureus and the type III Fc receptor, which is found on
certain strains of group C and group G streptococci. The scant amount
of information on the other bacterial Fc receptors is due in large part
to the failure to identify bacterial strains with high levels of stable
Fc receptors type II, IV or V on their surface.
The involvement of these Fc receptors in the pathogenicity of
disease is unknown. Protein A has been shown to activate complement
and cause B-cell nitogenesis in vitro and immediate-type hypersensitiv
ity reactions in vivo. Little is known, however, about the biological
properties of the streptococcal Fc receptors and whether they are


10
involved in the pathogenicity of disease. Sore evidence suggests that
there is a correlation between the virulence of certain group A
streptococci and their ability to bind the He region of human IgG.
Until the group A streptococcal Fc receptors are purified and the
biological activities explored, however, the importance of these
receptors in the pathogenesis of group A streptococcal infections, and
post-infection sequelae, will be difficult to assess.
The purpose of this study was to isolate and characterize the type
II Fc receptors found on certain yroup A streptococci and to examine
the distribution of the type II Fc receptors and their relationship in
the pathogenesis of streptococcal diseases. The specific aims were
to:
1. Develop a method to screen bacterial isolates for Fc receptor
proteins, in particular the type II Fc receptors (Chapter
Two).
2. Isolate and characterize the type II Fc receptors from group A
streptococci (Chapter Three).
3. Determine the distribution of the type II Fc receptors on
nephritogenic and non-nephritoyenic group A streptococci
(Chapter Four).


CHAPTER TWO
A TECHNIQUE FOR THE DETECTION OF BOUND AND SECRETED
BACTERIAL Fc RECEPTORS
Introduction
The type II Fc receptor on certain group A streptococci has proved
difficult to study because it is only found at low levels on positive
strains and is frequently lost during subculturing. The initial aim of
this study v/as, therefore, to establish a rapid method to screen group
A streptococcal strains for Fc receptors and to identify and expand
individual colonies that expressed high levels of these receptors.
Previous studies have shown that passage of streptococci in nice
resulted in enhanced Fc receptor expression (Burova et_ al_., 1980,
1981). This approach has been used to isolate a group A streptococcal
strain with increased Fc receptor expression (Reis et_ £]_., 1984d).
Until a method is developed to screen fresh isolates of group A
streptococci without subculturing, it will be difficult, however, to
assess the role that this receptor may play in group A streptococcal
infections and post-infection sequelae.
This chapter describes a semi-quantitative procedure that enables
isolates of group A streptococci to be screened rapidly for Fc receptor
expression. Using this technique, a mouse-passaged group A strepto
coccal strain that was selected for its high levels of surface Fc
receptors (Reis et aj_., 1984d) was shown to be heterogeneous in the
level of expression among individual colonies. Colonies expressing
high levels of surface Fc receptors were selected from replica plates
11


12
and were shown to maintain a higtier average level of Fc receptor
expression on repeated subculture.
Materials and Methods
Bacterial Strains, Media, and Growth Conditions
Laboratory strains and fresh isolates of p-henolytic streptococci,
obtained from Dr. El id Ayoub dt the University of Florida, College of
Medicine, and Staphylococcus aureus strains, obtained from the American
Type Culture Collection, were used in these studies, p-hemolytic
streptococci were grouped by the Phadebact Streptococcus Test,
Pharmacia Diagnostics, Piscataway, NJ. All strains were grown in
Todd-Hewitt broth (DIFCO) for 18-24 hr at 37C, harvested by
centrifugation, and washed in phosphate-buffered saline (PBS), pH 7.2.
The optical density at 55 mm was determined to standardize the
concentration of organisms used in subsequent tests.
Staphylococcus aureus Cowan I and Staphylococcus aureus Wood 46
served respectively as high and low protein A (PA)-producing strains.
IqG and F(ab)? Fragments
Stock human IgG was prepared by chromatography of normal human
serum on DEAE cellulose (Boyle and Langone, 1980). Aliquots viere stored
at -70C until use. Human IgG F(ab')2 fragments were prepared by
pepsin digestion of the stock IgG preparation by the method described
by Reis et aj_. (1983). The IgG and F(ab')2 fragments viere prepared
from an individual donor and therefore the IgG and F(ab')2 fragments
displayed the same distribution of antigenic reactive antibodies.
Iodination of IgG
Purified human IgG was iodinated by mild lactoperoxidase method
using enzyme beads (Bio-Rad) as described previously (Reis et al.,
1983). The IgG routinely had a specific activity of 0.3 mCi/mg.


13
Detection of Surface Bacterial Fc Receptors
An overnight suspension of bacteria was diluted in Todd-Hewitt
broth to give 1-10 colonies when 0.1 ml was plated on Todd-Hewitt
agar (Todd-Hewitt broth containing 1.5% agar). The plates were
incubated at 37C for 16 h and replica plated onto blood agar plates
(BBL Microbiology Systems, Cockeysville, MD 21030) as described by
Lederberg and Lederberg (1952). A circular piece of nitrocellulose,
previously soaked in 25 nM Tris, 192 rnM glycine (pH 8.3) and 20% v/v
methanol followed by a circular piece of Whatman 3 mn paper was placed
on top of the colonies. The agar was removed from the petri dish and a
piece of 3 mm paper was placed on the bottom of the agar. The
bacterial colonies were transferred to the nitrocellulose by
electrophoresis at 70 V for 3 h in the above buffer. This procedure
resulted in quantitative transfer of the bacterial colonies, as judged
by comparing photographs of plates before transfer with the stained
nitrocellulose membrane after the electroblotting procedure.
After electrophoresis, the nitrocellulose was washed in veronal
buffered saline (VBS) containing 0.25% gelatin and 0.25% Tween-20 for
one hour with four 250 ml changes. The nitrocellulose was probed for
3 h in the washing buffer containing 2 x 105 cpm/ml 125i_iabeled
human IgG and a two-fold excess of unlabeled F(ab')2 fragments.
Inclusion of F(ab)2 fragments in the probing mixture eliminates any
binding of IgG through antigenic recognition sites (Reis et_ al_., 1983).
After probing, the nitrocellulose was washed four times in 0.01 M EDTA,
1 fl HaCl, 0.25% gelatin, and 0.25% Tween-20 for 15 min each and allowed
to air dry. The nitrocellulose blots were autoradiographed by exposing


14
to Kodak XAR-5 film with intensifying screen for 3 days at -70C. This
procedure is summarized in Figure 2-1.
Absorption of 125i-Labeled IgG by Bacteria
The detection of Fc-reactive proteins on the surface of bacteria
was determined by the ability of bacteria to bind 125iabeled
human IgG using a modification of the method of Myhre and Kronvall
(1977). Briefly, a standard number of bacteria was incubated with
20,000 cpm 125j-labeled human IgG and a twofold excess of unlabeled
F(ab')2 fragments for 1.5 h at 37C. The bacteria were pelleted by
centrifugation at 1000 x g for 5 min and washed twice with 2 ml of VBS
containing 0.01 M EDTA and 0.1% gelatin (EDTA-gel). The radioactivity
associated with the bacteria was determined in an LKB autogamma
counter. The cpm bound to the bacterial pellet were expressed as
percent of added radioactivity.
Results
Fc Receptor Expression of Staphylococcus aureus
The purpose of this study was to develop a procedure that could
rapidly screen isolated colonies of bacteria for Fc receptor
expression. Initially I used the protein A-rich Staphylococcus aureus
Cowan I strain as a reference high level positive control and the
Staphylococcus aureus Wood 46 strain as a reference low level positive
control. In these experiments, a dilution of Staphylococcus aureus
Cowan I or Wood 46 strain was seeded onto Todd-Hewitt agar plates to
yield approximately 5 colonies following overnight incubation at 37C.
The colonies were transferred to nitrocellulose and probed with
^251_-| abe]e(j human IgG containing a two-fold molar excess of


15
unlabeled F(ab)2 fragments. The F(ab')9 fragments were prepared
from the same source of human IgG as that used to prepare the labeled
tracer. Previous studies have shown that inclusion of the F(ab')2
fragments in the probing mixture eliminates any binding of IgG through
antigenic recognition sites (Reis e^ aU, 1983). Consequently, only
binding via the Fc region is measured using this probing mixture. The
procedure is detailed in the Methods and summarized in Figure 2-1. The
results of a typical experiment using Staphylococcus aureus strains are
presented in Figure 2-2. The Staphylococcus aureus Cowan I strain
demonstrated a greater expression of Fc receptors than Wood 46 strain.
The surface expression of Fc receptors on an individual plate was
consistent from colony to colony, for both the Cowan I arid the Wood 46
strain.
Quantification of Secreted Fc Receptors
Secreted Fc receptors were measured using a modification of the
blotting procedure described in Figure 2-1. After removal of the agar
from the petri dish, the nitrocellulose was placed on the underside of
the agar away fron the bacteria rather than directly in contact with
the colonies. Fc receptors that were secreted by the bacteria were
electroblotted through the agar and bound to the nitrocellulose while
the colonies were unable to pass through the agar. The nitrocellulose
was probed as before with 125j human IgG containing unlabeled
F(ab')2 fragments, and then autoradiographed. Figure 2-2 illustrated
the secretion of Fc receptors by the Cowan I and Wood 46 strain. As
expected, the Cowan I strain secreted high levels of Fc receptor
activity. By contrast, the Wood 46 strain did not secrete sufficient
protein A to be detected by this procedure, see Figure 2-2.


16
I. REPLICA
PLATE
AGAR FROM
PETRI DISH
5. AUTO
RADIOGRAPH
Fig. 2-1. Schematic representation of the imunobl otti ng procedure for
detection of surface Fc receptors.


17
l2BI IgG Binding to Staphylococcal Strains
Cell Associated Secreted
v
B
Fig. 2-2. Detection of Fc receptors on the surface of staphylococcal
strains.
A, Staphylococcus aureus Cowan I strain; B, Staphylococcus aureus
Wood strain. The colonies of staphylococci were blotted onto
nitrocellulose and probed with 125j^abeled human IgG in the
presence of unlabeled F(ab')2 fragments as described in the text.
Autoradiography was carried out by exposure of the blot for 3 days at
-70C to X-ray film using an intensifying screen.


18
In order to determine the sensitivity of this method for detecting
secreted Fc receptors, differing concentrations of purified protein A
(0.05-500 ng/5 pi) were applied directly to agar plates. The agar was
removed and treated as described above. The applied protein A was
electroblotted through the agar onto nitrocellulose and probed for
binding of labeled 125i human IgG. The blot was autoradiographed
at -70C for one day and the diameter of the resulting dot was
measured. The area of the dot was proportional to the square of its
radius (r). When the value of r2 for each concentration of protein A
originally applied to the agar was plotted against the concentration of
protein A added, a linear relationship was observed over the range 6.25
to 50 ng, see Figure 2-3.
Fc Receptor Expression on a Group A Streptococcal Strain
In the initial experiments a mouse-passed strain of group A
streptococci, 64/14, which has been shown to have high levels of
surface Fc receptor (Reis et_ al_., 1984d), was selected to study the
expression of Fc receptors on the surface of streptococci. A single
colony of the mouse-passed strain 64/14 was grown overnight in
Todd-Hewitt Broth. Bacteria were then diluted as described in the
Methods to yield 10-10 colonies per plate and tested for Fc receptor
expression. The Fc receptor expression on the individual colonies of
strain 64/14 is illustrated in Figure 2-4. Unlike the uniform pattern
observed with Staphylococcus aureus strains (Figure 2-2), the intensity
of the spots on the autoradiograph varied considerably, indicating
heterogeneity in expression of Fc receptors on individual colonies.
This effect was not due to variation in the size of individual
colonies, since, as shown on the replica plated sample, the colony size


19
ng PROTEIN A ADDED
Fig. 2-3. Sensitivity of assay for secreted Fc receptors.
Protein A fror.i 0.5 to 500 ng was applied in 5 yl of VBS, to a
culture plate, electroblotted through the agar onto nitrocellulose and
then probed with 125i_iabeiec| human IgG in the presence of
unlabeled F(ab1)2 fragments. The blot was autoradiographed by
exposure for 1 day at -70C to X-ray film using an intensifying screen
and the diameter of the resulting dot on the X-ray film was measured.
The value of r2, which is proportional to the area of the dot, was
plotted against the concentration of protein A applied to the plate.


20
Fig. 2-4. Fc receptor expression of a group A streptococcal strain.
The panel on the left of the figure shows colonies of strain 64/14
on a blood agar replica plate. The panel on the right is an autoradi
ograph of a replica plate following the probing with 1251_iabeled
human IgG in the presence of unlabeled F(ab')2 fragments. Autoradi
ography was carried out by exposure of the blot for 3 days at -70C to
X-ray film using an intensifying screen.


21
was remarkably uniform. A representative high intensity (Fc receptor-
rich) and low intensity (Fc receptor-poor) colony was selected from the
replica plate and subcultured. The expression of surface Fc receptor
on the progeny of a 1ow-producer colony and a high-producer colony are
shov/n in Figure 2-5. The progeny of the high producing colony
maintained greater levels of Fc receptor expression than the progeny of
the low producing colonies; however, heterogeneity in surface
expression of Fc receptors within the selected strains was still
observed. On repeated subculturing, however, the low and high
producers remained readily distinguishable, and none of these strains
secreted measurable quantities of the Fc receptor.
The differences in average Fc receptor expression of these two
bacterial subpopulations were also tested in a direct binding assay.
In this assay the indicated number of bacteria was incubated at 37C
for 1.5 h with 2 x 104 cpm/0.1 ml 125¡ human IgG and a two-fold
molar excess of unlabeled F(ab*)2 fragments in a total volume of
0.2 ml VBS-gel buffer. The bacteria were pelleted by centrifugation at
1000 x g for 5 min and washed twice with 2 ml of EDTA-gel buffer and
the number of cpm bound to the bacteria determined. The results in
Figure 2-6 demonstrate greater IgG binding to the high producing
population than to the low producing population, with the parental
64/14 strain binding an intermediate level. These results are in
agreement with the findings in Figure 2-5 and would suggest that the
blotting assay is accurately reflecting surface Fc receptor expression
ori these bacteria.


22
Fiy. 2-5. Fe receptor expression of two bacterial subpopulations
selected iron the original strain 64/14.
The autoradiograph in the left panel was obtained by probing a
high Fc receptor producing substrain and the autoradiograph in the
right panel was obtained by probing a low Fc receptor-producing colony.
These colonies were selected and subcultured fron individual colonies
in the replica plate shown in Figure 2-4. Autoradiography was carried
out by exposure of the blot for 3 days at -70C to X-ray filn using an
intensifying screen.


Number of Bacteria
Fig. 2-6. Fc dependent uptake of 125jabeled hunan IyG by
increasing numbers of bacteria.
* *, parental strain 64/14; a a, high Fc receptor producing
substrain from 64/14; oo, low Fc receptor producing substrain from
64/14.


24
Discussion
In this chapter, a method for rapidly screening fresh isolates of
streptococci for Fc receptors has been described. This technique can
measure both cel 1-associated and secreted Fc receptors and is capable
of identifying individual colonies that express high levels of these
receptors. Initially, the method was developed using Fc receptor
positive Staphylococcus aureus strains that express stable levels of
the type I Fc receptor on their surface. These were the protein A-rich
Cowan I strain and the protein A-poor Wood 46 strain (Freimer et al,
1979; Reis et^al_., 1984c). The conditions for electroblotting
bacterial colonies onto nitrocellulose and probing for Fc receptor
expression were established using these strains (Fig. 2-2). The
^I-labeled human IgG probe was made Fc receptor-specific by
including a two-fold molar excess of unlabeled F(ab')2 fragments from
the same IgG pool used to prepare the labeled tracer. Under these
conditions the unlabeled F(ab')2 fragments inhibit the binding of any
specific anti-bacterial antibody (Reis et_ aj_., 1983). In agreement
with previous reports, all of the staphylococcal colonies expressed
surface protein A, and there was a marked quantitative difference
between the levels expressed on the Cowan and Wood strains (Freimer et
al ., 1979; Reis et_ a]_., 1984c).
When this approach was applied to a mouse-passaged group A
streptococcal strain, the intensity of individual autoradiographed
streptococcal colonies showed wide variation (Fig. 2-4). In order to
ensure that these findings were reproducible, one colony that expressed
high levels of Fc receptors and a second colony, with a low level of Fc
receptor expression, were subcultured from the replica plate.


25
Following subculture, each colony was plated, electroblotted and probed
as described. The results indicated that the intensity of the progeny
colonies were markedly different (Fig. 2-5). Differences in surface Fc
receptor expression of the two substrains were confirmed by direct
Fc-rnediated binding of labeled human Iyt (Fig. 2-6). In the two days
required to expand a high or low expressing population, considerable
heterogeneity in Fc receptor expression on the resulting colonies was
readily detected (Fig. 2-4). These findings suggest that the
expression of these type II Fc receptors on group A streptococci is
constantly shifting and could explain many of the reports of changes
in, or loss of, Fc receptor expression during subculture of group A
streptococcal strains (Kronvall, 1973a; Christensen and Oxelius, 1974;
Freimer et_ al_., 1979).
The presence of extrachronosonal DNA in certain group A strepto
cocci has been reported (Clewell, 1981; Burova et_ al_., 1983;
Ravdonikas, 1983; Ravdonikas et aj_., 1984). Conjugation experiments
between group A streptococcal strains with Fc receptors and group A
streptococcal strains without Fc receptors, suggest that either Fc
receptor expression is controlled by a plasmid, or that the gene for Fc
receptors is encoded on a plasmid (Burova et jil_., 1983; Ravdonikas et
al., 1984). No plasmids have been isolated, however, from the mouse-
passaged group A streptococcal strain 64/14 (data not shown). The
possibility still exists that the plasmid has integrated into the
genome, but more studies are needed in order to understand the control
of Fc receptor expression that is expressed on group A streptococcal
strains.


26
In addition to providing a rapid, semi-quantitative assay for
surface Fc receptor expression on individual colonies of bacteria, the
method described was readily modified to measure secreted Fc receptors.
This was achieved by carrying out the electroblotting stage of this
assay with the nitrocellulose placed ori the opposite side of the agar
from the bacterial colonies. The approach readily detected nanogram
quantities of secreted protein A from the staphylococcal strains (Fig.
2-3). To date, no Fc receptor secreting group A streptococcal strain
has been identified.
The method described in this chapter for screening individual
colonies of bacteria for Fc receptors has enabled me to monitor type II
Fc receptor expression on a group A streptococcal strain. By continual
selection, I can now maintain a strain rich in the type II receptor
from which to isolate this receptor.
This method can also be used to screen bacteria for other
receptors. Using a modification of the procedure P2-nicroglobulin,
fibrinogen, fibronectin, and collagen type I and type III receptors
were found on certain isolates of staphylococci and streptococci (see
Chapter Four). In addition, this method can be applied to the study of
fresh isolates of bacteria, to determine if the presence or absence of
a particular protein, or receptor, correlates with subsequent disease
course.


CHAPTER THREE
ISOLATION AND PARTIAL CHARACTERIZATION OF THE TYPE II Fc RECEPTORS
FROM A GROUP A STREPTOCOCCUS
Introduction
Although many group A streptococci have surface receptors for the
Fc region of all four subclasses of human IgG (Myhre and Kronvall,
1977,1979,1980b,1981; Reis et a]_., 1984d), these receptors are
frequently lost during subculturing (Christensen and Oxelius, 1974;
Freimer et aj_., 1979), making the isolation and characterization of
these proteins difficult. Recently Reis et_ aj_. (1984d) have described
the isolation of a stable Fc receptor-rich group A streptococcal
strain. This strain, which was recovered following fourteen sequential
passages of a group A streptococcus in mice (Reis et _al_., 1984d),
demonstrated Fc receptor expression approaching that of the protein A
rich Staphylcoccus aureus Cowan I strain (Reis jit jil_., 1984d).
Although it has maintained a high level of Fc receptor expression for
over two years, heterogeneity of expression was observed between
individual colonies when monitored using an immunoblotting assay
(Chapter Two). The immunoblotting technique has enabled me to monitor
Fc receptor expression continuously on individual colonies and to
select substrains with high levels of Fc receptor expression. In this
Chapter, I describe the isolation and characterization of the Fc
receptors from such an Fc receptor-rich group A streptococcal
substrain.
27


28
Materials and Methods
Bacterial Strains, Media, and Growth Conditions
The mouse-passaged group A streptococcal strain 64/14 was shown in
Chapter Two to contain colonies with different levels of surface
expression of Fc receptors. Using the immunoblotting technique
described in Chapter Two, a single colony expressing high surface Fc
receptor activity (64/14/HRP) was selected and used as the source for
the isolation of the type II Fc receptor. Staphylococcus aureus Cowan
I strain served as a representative type I Fc receptor-positive strain
and the group C streptococcal strain 26RP66 served as a type III
receptor-positive strain. All strains were grown in Todd-Hewitt broth
(UIFC) as stationary cultures for 18-24 hr at 37C, harvested by
centrifugation and resuspended in phosphate-buffered saline (PBS), pH
7.2. The optical density at 55 nm was determined to standardize the
concentration of organisms used in subsequent tests. An OD^^q of
U.3 corresponded to approximately 2 x 10^ organisms/ml.
Dipeptides
Glycyl-L-tyrosine, glycyl-L-histidine, and glycylglycine were
purchased from Sigma Chemical Company, St. Louis, MO.
Extraction of Fc Receptors
The selected group A streptococcal strain 64/14/HRP was grown
overnight at 37C in Todd-Hewitt broth. Approximately 6 g (wet weight)
of bacteria were recovered from 3 liters of Todd-Hewitt broth.
Mutanolysin extraction. Approximately 6 g (wet weight) of group A
strain 64/14/HRP were suspended in 30 ml of 20 mM Tris-HCl, pH 7.5, 1
mM iodoacetic acid, arid 1 mM benzamidine HC1. To this suspension, 100
ug/nl pancreatic DNase (Sigma), 100 yg/nl pancreatic RNase (Sigma), and


29
100 yg/ml mutanolysin were added. A commercial mutanolysin preparation
(Sigma) was further purified to remove protease activity using the
method described by Siegel jit aJL (1981). The enzyme and bacteria were
incubated at 37C in a shaking water bath for 4 hr. The mixture was
then centrifuged at 10,000 g for 10 min and the resulting supernatant
filtered through a 0.2 urn filter to remove the remaining bacteria. The
filtrate was dialyzed against 20 mM Tris-HCl, pH 7.5, containing 1 mM
iodoacetic acid, 1 mM benzamidine-HCl, and 1 mM phenylmethyl sulfonyl
fluoride (PMSF).
Hyaluronidase extraction. Approximately six grams (wet weight) of
the group A strain, 64/14/HRP, were suspended in 30 ml of 0.15 M PBS,
pH 7.2. To this suspension, 10 mg type IV hyaluronidase (Sigma) was
added and incubated at room temperature for 30 min. The bacteria-free
supernatant was recovered as described above.
Papain digestion. Group A strain 64/14/HRP, approximately 2 g
(wet weight), was suspended in 20 mis of 10 mM Tris-HCl, pH 8.0 and
0.02% NaN3. Two milliliters of 0.4 M cysteine and 1.6 mg papain were
added to this suspension and allowed to incubate at 37C for 1 hr. The
reaction was stopped by addition of iodoacetic acid to a final
concentration of 30 mM. The bacteria-free supernatant was recovered as
described above.
Trypsin digestion. Approximately 2 grams (wet weight) of
group A strain 64/14/HRP in 50 mM KPO4, 5 mM EDTA, 0.02% NaN3, pH
6.1 (20 ml) was incubated at 37C for 1 hr. with 80 yg pancreatic DNase
and 400 yg trypsin (Sigma). Addition of benzamidine-HCl to a final
concentration of 100 mM stopped the reaction. Bacteria were removed as
described above.


30
Phage lysin treatment. Approximately 2 g (wet weight) of the
group A strain 64/14/HRP were suspended in 20 ml of 50 mM KPO4, 5 mM
EDTA, 0.02% NaN3, pH6.1. Phage lysin (0.2 ml), previously activated
by incubation at room temperature for 15 min. in dithiothreitol (DTT)
at a final concentration of 50 mM, was added to the 10% suspension and
incubated at 37C for 1 hr. The phage lysin was prepared as previously
described by Fischetti et al_. (1971). The bacteria-free supernatant
was recovered as described above.
SDS treatment. A 10% (w/v) suspension of group A strain 64/14/HRP
in 1% sodium dodecyl sulfate was incubated at 80C for 10 min.
Bacteria were removed as described above and SDS was removed by
dialysis against 20 mM Tris-HCl, pH 7.5.
Autoclave treatment. A 10% (w/v) suspension of group A strain
64/14/HRP was autoclaved at 124C for 15 min, with or without the
addition of 1% SDS.
Hot acid/hot alkaline extractions. Extractions were carried out
according to the method of Lancefield (1928). Bacteria were suspended
in 0.15 M PBS to form a 10% suspension and the pH was adjusted to 2 (or
10) with 0.5 M HC1 (or 0.5 M NaOH). The bacterial suspension was
boiled for 10 min and the pH neutralized. The bacteria-free super
natants were recovered as described previously.
Heat extraction. This was carried out as described above, but at
neutral pH using PBS, pH 7.0.
IqG and IgG Subclasses
Stock human IgG was prepared by chromatography of normal human
serum on DEAE cellulose (Boyle and Langone, 1980). Aliquots were
stored at -70C until use. Human IgG3 (k) cryoglobulin was a gift


31
from Dr. Richard Weber, National Institutes of Health, Bethesda, MD.
The cryoglobulin was isolated as described in (Saluk and Clem, 1971).
Human IgG subclasses were provided by the WHO/IUIS Immunoglobulin
Subcommittee.
IgG} (k) Lot No. 0781 and IgG¡ (x) Lot No. 0180;
IgG2 (K) Lot No. 0380 and IgG2 (x) Lot No. 0981;
IgG3 {<) Lot No. 0282 and IgG3 (<) Lot No. 0784 and
IgG3 (X) Lot No. 0381;
IgG4 (K) Lot No. 0981 and IgG4 (x) Lot No. 0880
Purified rabbit, cow, sheep, goat, rat, dog, and pig IgG was purchased
from Cappel Laboratories, Inc., Cochranville, PA.
Immobilized IgG Preparations
Antigens were coupled to immunobeads (Bio-Rad) by the method
described in Reis et aj_. (1983). The ligand to be immobilized was
covalently bound to the immunobead matrix by peptide bond formation
between the carboxylic groups on the beads and amino acids groups of
the ligand. This reaction is catalyzed by carbodiimide and the
resulting beads can be stored for up to six months at 4C in the
presence of 0.02% sodium azide without loss of reactivity.
Immobilized IgG for affinity purification of the streptococcal Fc
receptor was prepared by covalently coupling human IgG to the high
capacity Affi-gel 15 activated beads (Bio-Rad) as described in (Reis et
al_., 1984b).
Iodination of IgG and Protein A
Purified Protein A (Pharmacia, Piscataway, NJ) or purified IgG or
IgG subclasses were iodinated by mild 1actoperoxidase method using
enzyme beads (Bio-Rad) as described previously (Reis et_ a1_., 1983).
The IgG or protein A routinely had a specific activity of 0.3 mCi/mg.


32
Preparation of Fc Specific Probe
125I labeled human IgG was made Fc specific by the inclusion
of a twofold molar excess of unlabeled F(ab')2 fragments in the
probing mixture (Reis et a]_. 1983). Human IgG F(ab')2 fragments
were prepared by pepsin digestion of the stock IgG preparation by the
method described by Reis et al. (1983). The IgG and F(ab')2
fragments were prepared from an individual donor and consequently the
IgG and F(ab')2 fragments contained the same distribution of
antigenic reactive antibodies. Therefore, only binding via the Fc
region is measured using this probing mixture.
Competitive Binding Assay For Soluble Bacterial Fc Receptor
Fc receptor activity in extracts were quantified using the
competitive binding assay of Reis et_ aj_. (1983). This assay was
carried out using VBS-gel as the diluent. In this assay 1.0 ml of a
test sample or buffer was mixed with 0.1 ml of a standard suspension of
agarose beads with covalently coupled human IgG (Bio Rad Laboratories,
Richmond, CA), and 0.1 ml of 125j protein A (approximately 20,000
cpm) and incubated at 37C for 90 min. Two milliliters of veronal
buffered saline containing 0.01 M trisodium ethylenediamine tetra
acetate and 0.1% gelatin (EDTA-gel) was added to each tube and
centrifuged at 1,000 g for 5 minutes and the supernatant fluid
decanted. After an additional wash, the radioactivity associated with
the beads was determined in an LKB Gamma Counter.
Gel LIectrophoresis and Western Blotting
Proteins were analyzed by electrophoresis under denaturing
conditions in polyacrylamide gels containing sodium dodecyl sulfate
according to Laennli (1970). Gels for staining were fixed in a


33
solution of 40% ethanol and 10% acetic acid, stained with Coonassie
brilliant blue R-250 (0.25% w/v in 40% ethanol and 10% acetic acid) for
1 hr, and destained by soaking in several changes of 10% ethanol and
10% acetic acid. Gels used for blotting were equilibrated in 25 nM
Tris, 192 rnM glycine, pH 8.3 and 20% v/v methanol (transfer buffer) for
30 min. A piece of nitrocellulose, previously soaked in the transfer
buffer, was placed on top of the gel. The gel and nitrocellulose were
sandwiched between 2 pieces of Whatman 3 mm paper and placed in a
Bio-Rad Trans Blot apparatus with the nitrocellulose oriented between
the anode and the gel. Electrophoresis was at 70 volts for 3 hr in the
above buffer.
After electrophoresis, the nitrocellulose was washed in veronal
buffered saline (VBS) containing 0.25% gelatin and 0.25% Tween-20 for 1
hr with four 250 ml changes. The nitrocellulose was probed for 3 hr in
the washing buffer containing 2 x 10$ cpm/ml of the appropriate
species or subclass of IgG. After probing the nitrocellulose was
washed four times in 0.01 M EDTA, 1 M NaCl, 0.25% gelatin, and 0.25%
Tween-20 for 15 min each and allowed to air dry. The nitrocellulose
blots were autoradiographed by exposing to Kodak XAR-5 film with
intensifying screen for 1 to 3 days at -70C.
Dot-Blot Procedure to Test Species Reactivity
Dot blots were performed using the Bio-Rad bio-dot microfiltration
apparatus and a modification of the Bio-Rad procedure. A standard
number of group A strain 64/lb/HRP was incubated at 37 for 1 hr with
increasing concentrations of dog, goat, pig, sheep, rabbit, rat,
bovine, or human IgG. A piece of nitrocellulose, previously soaked in
25 mM Tris, 192 nM glycine, pH 8.3, and 20% V/V methanol (wash buffer),


34
was placed in the apparatus. Following incubation, the mixture was
pipetted into the wells and washed with the above buffer. The
nitrocellulose was removed from the apparatus, washed and probed as
described above in the Western blotting procedure.
Preparation of Monospecific Antibodies to a Single Species of Affinity
Purified Fc-Reactive Material
Monospecific antibodies were prepared in chickens whose non-immune
IgG does not react with the Fc-receptor protein being studied. The
choice of a non-reactive host to immunize is important to avoid compli
cations with hypersensitivity and Arthus reactions (Gustafson et al.,
1968). Immunoglobulins were isolated from egg yolks as described
below. Eggs from a white Leghorn chicken were collected prior to
injection with isolated Fc-reactive protein. This provided a source of
pre-immunization IgG from an individual animal. This was used as a
control for later studies. The chicken was then injected with an
immunogen containiny approximately 50 yg of Fc-reactive material
intramuscularly or subcutanously in complete Freund's adjuvant. The
immunogen used was a single form of the Fc-receptor protein that was
isolated first by affinity purification by binding to, and elution from
a column of immobilized IyG, and then further purified by SDS
polyacrylamide gel electrophoresis. The gel was stained with Coomassie
blue, and a sinyle stained band was cut from the yel, emmulsified in
adjuvant and used- as the immunogen. The chickens were allowed to rest
for three weeks and then injected with approximately 50 yg of the
immunogen prepared as described above that had been emmulsified in
incomplete Freund's adjuvant. Eggs were collected from the chickens,
immunoglobulins were extracted, and the production of antibody was
monitored as described below.


35
Chloroform Extraction of Egg Yolks
Eggs from the chicken were extracted using a modification of the
procedure described by Aulisio and Shelokov (1967). Briefly, the yolks
were separated fron the albumin and adhering membrane, diluted with an
equal volume of PBS, and shaken several times. The suspension stood at
room temperature for 10 min. The extraction was repeated a total of 4
times before centrifugation at 10,000 g for 20 min. To the super
natant, an equal volume of chloroform was added and the mixture was
shaken at room temperature every 30 min for 2 hr before incubating at
4C overnight. The extraction was centrifuged at 5,000 g for 10 min.
The resulting clear supernantant was assayed for antibody production by
measuring the inhibition of 125I labeled human IgG binding to the
type II receptor-rich group A bacterial strain, 64/14/HRP.
Chicken Anti-Type I and Anti-Type III Antibodies
Monospecific antiserum to the staphylococcal type I Fc receptor
was prepared as described in (Rei s ^?t jfL 1984c). Monospecific
antiserum to the streptococcal type III Fc receptor v/as prepared as
described by Reis et al_. (1984a).
Immobilized Anti-Type II Fc Receptor
Antibodies raised against the affinity purified 38,000 dal ton
(type IIb) Fc receptor present in the affinity purified heat extract of
64/14/HRP was prepared as described above. This anti-type lib Fc
receptor antibody was isolated by chloroform extraction of the yolks of
eggs from immunized chickens. The antibodies were concentrated by
ammonium sulfate precipitation (40%) and covalently coupled to Affi-gel
15 activated beads (Bio-Rad) as described in (Reis et jil_., 1984a). The
immobilized anti-type IIb Fc receptor was used for the affinity
purification of the type II Fc receptors.


36
Purified Type I and Type III Fc Receptors
Purified type I Fc receptor (protein A) was obtained from
Pharmacia Fine Chemicals, Piscataway, NJ. The type III Fc receptor was
purified as described by Reis, et al_. (1985).
Results
Solubilization of The Type II Fc Receptor
A mouse-passaged group A streptococcal strain, 64/14/HRP, was
selected because of its high levels of surface Fc receptors (Chapter
Two) and its stability on subculture (Reis et al_., 1984d). Several
extraction procedures were compared, including: 1) hot-acid
extraction, alkaline extraction, or heat extraction at neutral pH;
2) treatment with the enzymes hyal uronidase, mutanolysin, papain,
trypsin, or phage lysin; or 3) heating, or autoclaving the bacteria in
the presence of sodium dodecyl sulfate (SDS). The resulting cell-free
extracts were tested for soluble Fc receptor activity using two assays.
The first was a competitive binding assay, that measures the inhibition
of labeled protein A (the type I Fc receptor) to immobilized
human IgG and is capable of detecting nanoyram quantities of
Fc-reactive proteins (Reis et al_., 1983). The second assay was a
Western blotting procedure. In this technique, the extractions were
electrophoresed on an SDS polyacrylamide gel, electroblotted onto
nitrocellulose, and probed with the labeled human IgG Fc
specific probe. By running a duplicate gel and staining with Coomassie
brilliant blue, Fc receptor activity can be matched to specific protein
bands. The Western blottiny procedure has a number of advantages.
First, it enabled size heterogeneity of Fc receptors to be detected and
second, it detected Fc reactivities with sites on IgG not related to


37
the binding site for staphylococcal protein A. Details of these
procedures are described in the Materials and Methods.
The only treatments that resulted in the solublizatiori of
significant quantities of Fc receptor activity were heat extraction
at neutral pH, and treatment with the enzymes mutanolysin or
hyaluronidase (Table 3-1). Extraction with SDS, or autoclaving in the
presence of SDS, also resulted in solubilizing Fc receptor activity,
but with lower specific activity. Extraction of the group A
streptococci with acid, alkali, papain, trypsin, phage lysin, or by
autoclaving did not solubilize detectable quantities of a functionally
active Fc receptor in either assay. Comparison of the extracts by
Western blot analysis showed that the extract with the least
heterogeneity resulted from heat i ng t fie bacteria at neutral pH (Fig
3-1). This extraction procedure was consequently used to isolate the
type II Fc receptor.
Isolation of the Type II Fc Receptor
The type II Fc receptor-rich, group A streptococcus was heat
extracted as described in the Methods. The bacteria-free extract was
dialyzed against 0 r,M Tris-HCl, pH 7.5 containing protease inhibitors
at a concentration of 1 inf 1 phenyl methyl sulfonyl fluoride (PMSF), 1 mfl
iodoacetic acid, and 1 mil benzamidine-HCl to prevent degradation of the
Fc receptors (Grubb et al., 1982).
The dialyzed extract was applied to a column of human IgG
immobilized on Affi-gel 15 which had been prewashed with 3 M MgC12
and then equilibrated in 20 r.fl Tris-HCl, pH 7.5, containing protease
inhibitors. The extract was applied to the column, and unbound
material was eluted by washing with the Tris-HCl buffer. Fc receptor
was eluted by addition of 3 M MgCl2 to the 20mM Tris-HCl, pH 7.5


TABLE 3-1
Fc Receptor Activity in Streptococcal Extracts
Extraction
Yield
(yq/q bacteria)
Western Blot Analysis
(Number of protein bands)
Competitive Binding Assay
(uq required for 50% inhibiton)
Heat
36
2
1.5
Hot acid
--
None
No inhibition
Hot alkaline

None
No inhibition
Hyaluronidase
24
4
8.4
Mutanolysin
115
>0
0.2
Papain
--
None
No inhibition
Trypsin
--
None
No inhibition
SOS

7
>200
Autoclaving
--
None
No inhibition
Autoclaving + SDS
--
5
>200
Phage lysin

None
No inhibition


39
I 2 3 4 5 6
56k-
38 k-
Fig. 3-1. Western blot autoradiograph of affinity purified extractions
of 64/14/HRP.
Lanes 1 and 2 contain 1.8 yg and 4.2 yg of heat extracted Fc
receptor. Lanes 3 and 4 contain 4 yg and 12 yg of hyaluronidase
extracted Fc reactive material. Lanes 5 and 6 contain .5 yg and 1.2 yg
mutanolysin extracted Fc receptor. The affinity purified samples were
electrophoresed on an SDS polyacrylamide gel, electroblotted onto
nitrocellulose, and probed with pc specific probe as described
in Materials and Methods. Autoradiography was for 20 hr at -70C with
an intensifying screen.


40
buffer. Other eluting buffers, including 1% SDS solution, 0.1 M
glycine-HCl, pH 2.0 or 0.2 M glycyl tyrosine, were tested, but only
MgCl2 resulted in the recovery of significant quantities of
functionally active Fc receptors.
After elution from immobilized IgG, each fraction was dialyzed
overnight against PBS containing 10 rfi EDTA to facilitate the removal
of Mg++ and all samples were finally dialysed into PBS without
EDTA. Each fraction was assayed for Fc receptor activity using a
competitive binding assay. Fractions containing Fc receptor activity
were pooled and concentrated by Ami con Ultrafiltration using a PM-10
membrane with a molecular weight cut off of 10,000 daltons. Aliquots
were stored at -70C and maintained their Fc receptor activity for at
least 1 yr.
The size heterogeneity of the affinity purified Fc receptor
was determined by electrophoresis on an SDS polyacrylamide gel followed
by staining with Coomassie brilliant blue (Fig. 3-2A). Two protein
bands were observed. The major protein band had a molecular weight of
56,000 daltons and a minor band was observed at 38,000 daltons. To
determine if both bands had Fc receptor activity, a parallel SDS
polyacrylamide gel was run, the separated proteins were electroblotted
onto nitrocellulose and probed with the 125j labeled Fc specific
probe as described in Materials and Methods. The results of the
Western blot demonstrated that the 56,000 dal ton protein reacts
strongly with the 125j labeled Fc specific probe, whereas the
38,000 dalton protein demonstrated only weak reactivity (Fig. 3-2B).


41
I
2
B
, 2
38KX
Fig. 3-2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
the type II Fc receptor.
Panel A: Lane 1 contains 5 yg of the type I receptor (staphylo
coccal protein A). Lane 2 contains 4 ug of the type II Fc receptor.
Samples were electrophoresed under denaturing conditions and stained
with Coomassie brilliant blue as described in Materials and Methods.
Panel B is the autoradiograph of a duplicate nel which was electro-
blotted onto nitrocellulose and probed with!25i Fc specific probe.
Autoradiography was for 2 days at -70C using an intensifying screen.
Lane 1 contains 30 ng of the type I receptor. Lane 2 contains 4000 ng
of the type II receptor.


42
The Effect of Pi peptides on the Binding of IgG Subclasses to the Type
II Fc Receptor
Certain amino acid residues in the Fc region of human IgG have
been reported to be important in the bindiny of human IgG or certain
human IgG subclasses to the type I Fc receptor (protein A). Tyrosine
residues in the Fc region of human IgG (Ueisenhofer et_ al_., 1978) and
in protein A (Sjoholm et a]_., 1973) have been shown to be involved in
this interaction. The ability of protein A to bind certain allotypes
of IgG3 has been shown to be associated with a single amino acid in
the heavy chain (Recht et_ a]_., 1981; Haake et_ al_., 1982; Shimizu et
al, 1983). Comparison of amino acid sequences of immunoglobulins that
bind protein A with those that do not, has implicated the importance of
histidine residue at position 435 of the heavy chain of the IgG3
molecule (Haake et al_., 1982). Furthermore, Bywater (1978,1983)
reported that glycyl-tyrosine could elute human IgG bound to a protein
A sepharose column. When glycyl-tyrosine was tested for its ability
to displace the bound type II Fc receptor from a human IgG affinity
column, no Fc receptor activity was eluted. Glycyl-tyrosine and a
second dipeptide, glycyl-histidine, however, did inhibit the binding of
certain human IgG subclasses to the type II receptor-rich group A
strain 64/14/HRP (Fig. 3-3). The binding of human subclasses 1, 2 and
4 to 64/14/HRP was inhibited by both glycyl-tyrosine and glycyl-
histidine, whereas the effect of these two dipeptides on the binding of
IgG3 was minimal (Fig. 3-3). Glycylglycine had no effect on any of
the subclasses and none of these dipeptides inhibited the uptake of
labeled human IgG to the type I Fc receptor-positive Staphylococcus
aureus Cowan strain, or to the type III Fc receptor-positive group C
streptococcal strain 26RP66 (Fig. 3-4).


43
ID
O
03
(O
CO
<
-J
o
CD
ID
CO
100 200 300 400 100 200 300
DIPEPTIDE CONCENTRATION (mM)
400
Fig. 3-3. The effect of dipeptides on the binding of 125j_iabeled
human IgG subclasses to an Fc receptor-positive group A
streptococcus.
A standard number of group A streptococci was incubated for 1.5
hours at 37C with 20,000 cpm of the appropriately 125j-label ed
human IgG subclasses in the presence of varying concentrations of
dipeptide. The bacteria were pelleted by centrifugation at 1000 x g
for 10 min and washed twice with 3 ml of veronal buffered saline
containing 0.01 M EDTA and 0.1% gelatin. The radioactivity associated
with the bacteria was determined in an LKB autoyamma counter. The cpm
bound to the bacterial pellet were expressed as percent of maximum cpm
bound.
Panel A represents the results obtained fron the binding of 1251 XgG^
Panel B represents the results obtained from the binding of 125j igG2
Panel C represents the results obtained fron the binding of 125j igf^
Panel D represents the results obtained from the binding of 125j igG^
Glycylglycine
Glycyl-L-histidine
AA Glycyl-L-tyrosine


44
Fig. 3-4. The effect of glycyl-histidine on the binding of 125j_
labeled human IgG to three bacterial strains.
A standard number of Staphylococcus aureus Cowan I strain (*-*),
group C streptococcal strain 26RP66 (-), or the group A streptococcal
strain 64/ 14/HRP (-) was incubated for 1.5 hr at 37C with 20,000 cpn
of 125x labeled human IgG in the presence of the indicated amounts
of gycyl-histidine. The bacteria were pelleted by centrifugation at
1000 x g for 10 nin and washed twice with 2 nl of veronal buffered
saline containing 0.01 M EUTA and 0.1% gelatin. The radioactivity
associated with the bacteria was determined in an LKB autoganna
counter. The cpn bound to the bacterial pellet were expressed as
percent of maximum cpn bound.


45
Reactivity of the Type II Fc Receptor With Hunan IgG Subclasses
The differences in the observed reactivity between the human IgG
subclasses and the dipeptides (Fig. 3-3) raised the possibility that
more than one Fc receptor existed on the surface of the group A strain
64/14/HRP. To test this possibility, samples containing the affinity
purified heat extracted type II Fc receptor were electrophoresed on SDS
polyacrylamide gels, transferred to nitrocellulose by electroblotting
and parallel gels were probed with 125j labeled human IgG^,
IgG2, IgG3, or IgG4. The type I Fc receptor and the type III Fc
receptor were included on each gel as reference positive controls (Fig.
3-5).
The type I Fc receptor, protein A, contained one major protein
band with a Mr of approximately 52,000 that bound IgG^, IgG2, and
IgG4 but not Igfi3 (Fig. 3-5). The heat extracted, affinity
purified type II receptor contained a major protein band with an Mr
of approximately 56,000, and a minor band with a Mr of approximately
38,000. The major 56,000 dalton band reacted with human IgG^
IgC¡2, and IgG4 but failed to react with human IgG3. The minor
38,000 dalton protein reacted only with human 1903. The type III Fc
receptor demonstrated a single protein band with a Mr of
approximately 40,000 and this protein reacted with all four human IgG
subclasses (Fig. 3-5).
A major concern with the results obtained in Figure 3-5, was the
possibility that the observed reactivity of the type II receptor with
I9G3 was unique to the labeled myeloma probe being used. Conse
quently these studies were repeated using two other human IgG3
myeloma proteins. The results shown in Figure 3-6 indicate that all


46
Fig. 3-5. Human imnunoglobul in G subclass reactivity of bacterial Fc
receptors.
Lane 1 contains 20 ng of the type I reqeptor (Staphylococcal
Protein A). Lane 2 contains 100 ng of the type III Fc Receptor, and
Lane 3 contains 4,000 ng of the type II Fc Receptor. Samples were
electrophoresed under denaturing conditions in polyacrylamide gels
containing sodium dodecyl sulfate according to Laenmli (13). Gels were
equilibrated in 25inf'l tris, 192 mM glycine, pH 8.3 and 20% v/v methanol
(transfer buffer) for 30 min. A piece of nitrocellulose previously
soaked in the transfer buffer was placed on top of the gel. The gel
and nitrocellulose were sandwiched between 2 pieces of Whatman 3mm
paper and placed in a Bio-Rad trails blot appparatus with the nitro
cellulose oriented between the anode and the gel. Electroblotting was
at 70 volts for 3 hours in the above buffer. After electroblotting,
the nitrocellulose was washed in veronal buffered saline containing
0.25% gelatin and 0.25% Tween-20 for 1 hour with four 250 ml changes.
The nitrocellulose was probed for 3 hours in the washing buffer
containing 2X105 cpm/ml 125i human IgG of the appropriate
subclass. After probing the nitrocellulose was washed four times in
0.01M EDTA, 1M NaCl, 0.25% gelatin, and 0.25% Tween-20 for 15 min each
and allowed to air dry. Autoradiography was carried out by exposure of
the blot for 3 days at -70C to Kodak XAR-5 film using an intensifying
screen.
Panel A is the result from blots probed v/ith 1251 IgG^.
Panel B is the result from blots probed with 125i igQg.
Panel C is the result from blots probed with 12!31 lgG3.
Panel D is the result from blots probed with 125¡ igG4.


47
ABC
12 3 12 3 12 3
Fig. 3-6. Reactivity of three hunan myeloma IgG3 samples with
bacterial Fc receptors.
Lane 1 contains 20 ng of the type I receptor. Lane 2 contains 100
ng of the type III receptor and Lane 3 contains 4,000 rig of the type II
receptor. Samples were electrophoresed on parallel SDS polyacrylamide
gels, electroblotted onto nitrocellulose, and each blot was probed with
a different 125j labeled myeloma IgG3 protein as decribed in
the legend to Fig. 1. Autoradiography was carried out by exposure of
the blot for 24 hours at -70C to x-ray film using an intensifying
screen.
Panel A represents a blot probed with 125x lgG3 (k) Lot No. 0784.
Panel B represents a blot probed with 1251 ¡gQ^ (K) |_ot No. 0282.
Panel C represents a blot probed with 125i igG3 (a) Lot No. 0381.


48
three human IgG3 samples reacted with the minor Mr 38,000 protein
in the affinity purified heat extracts of the group A streptococcus.
One of the IgG3 samples was found to react with the type I receptor
(staphylococcal protein A) and this particular sample of IgG3 also
reacted with the major Mr 56,000 protein as well as the minor Mr
38,000 protein in the extract of the group A streptococcus (Fig. 3-6,
Panel A).
The IgG3 reactivity was further confirmed in inhibition studies
using unlabeled subclass standards. Only the sample containing IgG3
could efficiently inhibit the binding of the 125j labeled human
I9G3 to the type II receptor-positive group A streptococcus, while
equimolar concentrations of IgG^, IgG2 or IgG4 showed minimal
inhibition (Fig. 3-7). All of the samples of unlabeled IgG3 tested
were capable of inhibiting the binding of the labeled IgG39 with the
most efficient inhibitor being the sample that was also used to prepare
the labeled probe.
Separation of Two Functionally Distinct Type II Fc Receptors
The 56,000 dalton Fc receptor which reacted with IgG^, IgGg,
and IgG4 (designated type 11a), was separated from the 38,000 dalton
IgG3-specific Fc receptor (designated type 11b) by use of an
immobilized column of IgG3. Heat extracts of 64/14/HRP containing
both Fc receptors were applied to a column of an isolated human IgG3
(k) myeloma immobi 1 ized on Affi-gel 15. The column was pre
equilibrated in 20 mf1 Tris-HCl, pH 7.5. The type I la Fc receptor
failed to bind to the IgG3 column and was recovered in the void
volume of the column by washing with the Tris-HCl buffer. The type lib
receptor, which bound to the IgG33 column, was eluted with 3 M


49
Fig. 3-7. Inhibition of binding 125i labeled IgG3 to a group A
streptococcus by unlabeled human IgG subclasses.
Approximately 1 x 107 of the Fc receptor-rich group A
streptococci, were incubated at 37C for one hour with the indicated
amounts of each human IgG subclass. Following incubation, each mixture
was dotted onto a piece of nitrocellulose previously soaked in 25 mM
tris, 192 mi-1 glycine, pH 8.3 and 20% v/v methanol using the Bio-rad
bio-dot microfiltration apparatus. After each well was washed with the
above buffer, the nitrocellulose was removed and washed in veronal
buffered saline containing 0.25% gelatin and 0.25% Tween-20 (wash
buffer) for one hour with four 250 ml changes. The nitrocellulose was
probed for three hours in the wash buffer containing 2 x 105 cpm/ml
125i labeled human IgG3. After probing the nitrocellulose was
washed four times in O.Olfi EDTA, 1M Na Cl, 0.25% gelatin and 0.25%
Tween-20 and allowed to air dry. The nitrocellulose was cut into
sections that contained an individual we11 and the cpm associated with
each section of nitrocellulose was determined using an LKB autogamma
counter and the percent inhibition of 125j ^3 binding to the
bacteria was calculated.
IgGl
aa IgG2
0--0 IgG3
* IgG4


50
NgCI2 in 20 nM Tris-HCl, pH 7.5, dialyzed, and concentrated as
described above.
The unbound material from the IgG3 column (the type I la
receptor) was further purified by binding to, and eluting from a column
of immobilized human IgG. The affinity purified Fc receptors were
analyzed by the Western blotting procedure. The type I la and type 11b
Fc receptors were electrophoresed on SDS polyacrylamide gels,
electroblotted onto nitrocellulose and parallel gels were probed with
either 125¡ labeled human IgG to detect the type I la Fc receptor,
or 1251 labeled human IgG3 to detect the type 11b Fc receptor
(Fig 3-8). 125¡ labeled human IgG was used as a probe for the type
I la Fc receptor because of the distribution of subclasses 1, 2, and 4
in relation to subclass 3 found in normal human serum. Human IgG
subclass 3 comprises only 8" of the total human IgG in the serum
(Lewis et a]_., 1970). Therefore, the contribution made by IgG subclass
3 in the labeled human IgG probe is minimal.
In Fig. 3-8A, the type I la Fc receptor reacted strongly with the
human IgG probe and no contamination with the type 11b Fc receptor was
seen on the gel probed with 125j labeled IgG3 (Fig. 3-8B).
Similarly, the isolated type 11b Fc receptor reacted strongly with the
IgG3 probe (Fig. 3-8B) and no contamination of the type I la Fc
receptor was seen (Fig. 3-8A). The type I and type III Fc receptors
were included on each gel as reference positive controls.
Species Immunoglobulin Reactivity With the Type 11 a and Type 11b Fc
Receptors
Five distinct bacterial Fc receptors have been classified based on
the reactivity of whole bacteria with different sources of


51
Fig. 3-8. Separation of the type I la Fc receptor from the type 11b Fc
receptor.
Lane 1 contains 30 ng of the type I Fc receptor (protein A). Lane
2 contains 30 ny of the type III Fc receptor. Lane 3 contains 3 yg of
the type I la Fc receptor, and Lane 4 contains 2 yg of the type lib Fc
receptor. Samples were electrophoresed on SDS polyacrylamide gels,
electroblotted onto nitrocellulose, and probed with either 125x
labeled human IyG or IcjG3. Panel A represents a blot probed with
1^1 human IgG. Panel B represents a blot probed with 125
human IgGg.


52
immunoglobulins (Myhre and Kronvall, 1981). In this classmfation,
the type II Fc receptor associated with group A streptococci Should
bind all four subclasses of human IgG, and IgG from rabbit ed pig
(Myhre and Kronvall, 1977) (see Table 1-1). In the next-serf& of
experiments, the IgG species binding profile for the group A strain
64/14/HR P which was used to isolate the type I la and type Ilfr^c
receptors was determined. A standard number of group A str''ifi
64/ 14/HRP was incubated with increasing concentrations of txf, goat,
sheep, rat, pig, rabbit, bovine, or human IgG. After incubatFflan, the
mixture was dotted onto nitrocellulose and the nitroceTltfds^^s then
probed with either 125j labeled human IgG, or 125I labeled ei c
human IgG3. Details are described in Materials and Methods. %ie
results indicate that the Fc receptors on the surface of" groupd1A strain
64/14/HRP could only bind pig, rabbit, and human IgG. Dogv'-gbVt,
sheep, rat or bovine IgG could not inhibit the binding of lZB'l J
labeled human IgG (Fig. 3-9A) or 125l labeled human IgG3'(Ffg. ;
3-9B) to strain 64/14/HRP. v ra i n
The ability of pig and rabbit IgG to inhibit the binding bfc both
i-2^I labeled human IgG and 1251 labeled human IgG3 indicates1 nu:
that either the type I la arid type 11 b Fc receptors on the'surface of
group A strain 64/14/HRP are both capable of reacting, or fcortVPFt
receptors are linked.on the bacterial surface and the bindfrlg-human,
pig, or rabbit IgG to one Fc receptor sterically hinders the ability of
the other Fc receptor to bind. c re<;
The purpose of the next experiment was to determine if bottf'the
type I la and type 11 b Fc receptors reacted equally with rabbi t'-^nd pig
IgG, or if the inhibition seen in Fig. 3-9 with both 125i labeled
human IgG and human IgG3 was due to steric hindrance. ~ nu:


53
A
IgG ADDED (pg)
GOAT
PIG
DOG
RABBIT
SHEEP
RAT
COW
HUMAN
ro
O
(O
Q
m
C\J
in
OJ
d
in
d
d
d
d
d
o
c\j
o
o
o
d
d
c\j
CD
CD
ro






'£

#
* *
*


B
IgG ADDED (pg)
ro
O
<0
d
in
CVJ
in
CVJ
d
m
d
d
d
d
d
O
c\j
d
d
d
d
d
d
:
cvi
CD
CD
ro
GOAT
PIG

#

DOG
RABBIT


SHEEP
RAT
COW
HUMAN











Fig. 3-9. Inhibition of 125x human IgG or 125j human IgG3 to
group A strain 64/14/HRP by immunoglobulin G from a variety
of mammalian species.
A standard number of group A strain 64/14/HRP was incubated at
37C for 1 hr with the indicated amount of goat, pig, dog, rabbit,
sheep, rat, cow, or human IgG. Following incubation, each mixture was
dotted onto nitrocellulose. The nitrocellulose was washed and probed
with 125j labeled human IgG or human IgG3 as described in
Materials and Methods. Autoradiography was for 16 hr at -70C with an
intensifying screen.
Panel A represents the blot probed with 125j labeled human IgG.
Panel B represents the blot probed with ^25j labeled human IgG3.


54
Samples containing both the type I la and type lib Fc receptors
were electrophoresed on SDS polyacryladmide gels, electroblotted onto
nitrocellulose and probed with 125j labeled hunan IgG, pig IgG,
raDbit IgG, dog IgG, or bovine IgG (Fig. 3-1U). The results show that
only the type I la Fc receptor could bind 1251 labeled pig or rabbit
IgG. Reactivity with the type 11b Fc receptor was only seen with
125i labeled human IgG (Fig. 3-10). This suggests that the two Fc
receptors must be closely linked on the surface of group A strain
64/14/HRP to account for the partial inhibition of 125j labeled
IgG3 that is seen in Fig. 3-9B.
Antigenic Relationship Between the Type I la and Type lib Fc Receptors
In order to test whether both the type I la and type 11b Fc
receptors were antigenically related, antibodies were prepared in
chickens against the type I la and type 11b Fc receptors. The affinity
purified heat extract, containing both Fc receptors, was electro
phoresed on an SDS polyacrylamide gel and both the 56,000 dal ton Fc
receptor and 38,000 dal ton Fc receptor were identified by Coomassie
blue staining. Each band was cut from the gel and used as the
immunogen to prepare the monospecific antibodies using the immunization
schedule described in Materials and Methods. Antibody was isolated
from the egg yolk by chloroform extraction and activity was monitored
by the ability of the isolated extract to inhibit binding of 125j
hunan IgG or human IgG3 to the group A strain 64/14/HRP. The results
in Fig. 3-11A indicate that the antibody prepared against the type I la
(Mr = 56,000) Fc receptor can efficiently inhibit the binding of both
125i labeled human IgG and 125j labeled hunan IgG3 to the
group A strain 64/14/HRP. A similar result was observed using the


55
- Fiy. 3-10. Reactivity of IgG from a variety of species with the type
I la or type 11b Fc receptor.
Affinity purified heat extract (4 pg) containing both the type I la
and type lib Fc receptors was electrophoresed on five parallel SDS
polyacrylamide gels, electroblotted onto nitrocellulose and probed with
labeled human IgG (Lane 1), pig IgG (Lane 2), rabbit IgG (Lane
3), dog IgG (Lane 4), or bovine IgG (Lane 5) as described in Materials
and Methods. Autoradiography was for 30 hr at -70C using an
intensifying screen.


56
anti-type IIb Fc receptor (Fig. 3-1IB). No inhibition was observed
when normal chicken immunoglobulins were added. The shapes of the
inhibition curves of the IgG binding or the IgG3 binding were similar
for each antibody (compare Figures 3-11A and 3-11B). This finding
suggests that either each Fc receptor was present at an approximately
equal density and recognized with equivalent affinity, or that the two
Fc receptors were closely linked on the bacterial surface. When the
heat extract was affinity purified by binding to a column of
immobilized chicken anti-type lib Fc receptors and then eluting with 3
M MgCl2, both the type I la and type lib Fc receptors were recovered
in a functionally active form (data not shown). This provides evidence
that the two distinct Fc receptors are antigenically related and that
the observed inhibition by the anti-type I la and type lib antibodies on
the intact group A organism could not solely be attributed to steric
hindrance.
Antigenic Relationship of the Type I, Type II and Type III Fc
Receptors
The antigenic relationship between the type I, II and III
bacterial Fc receptors was tested using monospecific chicken antibodies
to each receptor type. Each antibody was tested at a number of
concentrations for its ability to inhibit the binding of
labeled human IyG to a fixed concentration of the group A streptococcal
strain 64/14/HRP. The results, presented in Fig. 3-12, indicate that
only the antibody against the type II Fc receptor could inhibit the
binding of labeled human IgG to strain 64/14/HRP. Antibodies
against the type I Fc receptor could not inhibit the binding of
labeled human IgG to the group A strain 64/14/HRP or to the


Fig. 3-11. Inhibition of binding of 125j_]abeled human IgG or
125i_iabeled human IgG subclass 3 to group A
streptococcal strain 64/14/HRP by monospecific antibodies
against the purified type I la Fc receptor or the type lib
Fc receptor.
A standard number of group A streptococcal strain 64/14/HRP was
incubated 1.5 hr at 37C with 20,000 cpm of 125j labeled human IgG
or labeled human IgG3 in the presence of the indicated
amounts of the monospecific chicken anti-type 11 a (Panel A) or
anti-type lib Fc receptor (Panel B). The bacteria were pelleted by
centrifugation at 1000 x g for 10 min and washed twice with 2 ml
veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin. The
radioactivity associated with the bacteria was determined in an LKB
autogamma counter and the percent inhibition calculated,
oo 125j labeled human IgG3
125j labeled human IgG


NOIligiHNI % NOI1IBIHNI %
58
RELATIVE ANTI-TYPE Hb Fc RECEPTOR


59
Fig. 3-12. Inhibition of binding of 125j human IgG to 64/14/HRP by
antibody against the type I receptor (-), the type II
receptor (-), or the type III receptor (o-o).
A relative antibody concentration of 10U for the type I antibodies
inhibits the binding of 125j human IgG to the Staphylococcus aureus
Cowan I strain by 94% and inhibits the binding of 1^5 ¡ human IgG to
the group C streptococcal 26RP66 strain by less than 10%.
A relative antibody concentration of 100 for the type III antibody
inhibits the binding of 125[ human IgG to the Staphylococcus aureus
Cowan Strain by less than 10% and inhibits the binding of
human IgG to the group C streptococcal 26RP66 strain by 92%.


60
group C Fc receptor-rich strain 26PP66, but could completely inhibit
the binding of the labeled immunoglobulin to the Staphylococcus aureus
Cowan strain. Similarly, the antibody to the type III receptor failed
to inhibit binding of hunan xgQ to either the group A
streptococcal strain 64/14/HRP or to the Staphylococcus aureus Cowan
strain v/hile totally inhibiting the binding of labeled immunoglobulin
to the Fc receptor-rich group C streptococcus 26RP66. No inhibition of
binding was observed to any bacterial strain when normal chicken
immunoglobulins were added.
Piscussion
In this Chapter I describe the isolation and characterization of
two functionally active Fc receptors from an Fc receptor-rich substrain
of a group A streptococcus. This bacterial substrain has been selected
from a mouse-passaged group A strain by use of an immunoblotting
technique that measures Fc receptor expression ori individual colonies
(Chapter Two). Several methods for solubilizing the Fc receptor from
this bacteria were tested, including: 1) heat extraction at neutral,
acid or alkaline pH, 2) digestion with the enzymes mutanolysin,
hyaluronidase, papain, trypsin, or phage lysin, or 3) autoclaving or
heating the bacteria in the presence of SDS. Soluble Fc receptor
activity was detected after heat extraction at neutral pH, treatment
with mutanolysin or hyal uronidase, or following autoclaving or heating
in the presence of SDS, Table 3-1. Mo activity was recovered following
any of the other extraction procedures (see Table 3-1). Heat
extraction at neutral pH resulted in the most homogeneous form of the
receptor, (see Fig. 3-1) and this also had one of the highest yields of
any of the extraction procedures tested (see Table 3-1).


61
Functionally active Fc receptor activity could be isolated from
the heat extract by binding to and elution from a column of immobilized
human IgG. A variety of different agents viere tested for their ability
to remove the bound receptor from the immobilized human IgG column and
the most efficient was found to be 3 M MgClg. Analysis of the
affinity purified material by SUS polyacrylamide gel electrophoresis
revealed two bands with molecular weights of 56,000 and 38,000 daltons
(Fig. 3-2). When the proteins were electroblotted onto nitrocellulose
and probed with a 125¡ human IgG Fc specific probe, both bands viere
shown to be active, although the 38,000 dal ton band had much less
activity. These two proteins bands could be shown to be antigenically
related, since both were recovered when the heat extract was affinity
purified on a column of immobilized chicken antibody that was directed
against a single form of the affinity purified Fc receptor. Although
these findings would be consistent with the 38,000 dal ton protein being
a degradation product of the larger 56,000 dal ton receptor, protease
inhibitors were included in all buffers used during the purification.
Another possibility was that these two molecular weight forms were
distinct Fc receptors. Recent studies by Wagner et al. (1983) using
immunoelectronmicroscopic approaches suggest that several distinct Fc
receptors can be present on the surface of a single group A
streptococcus. Analysis of the effects of various dipeptides (Fig.
3-3) and the reactivity of different human IgG subclasses (Fig. 3-5)
confirmed the existence of at least two distinct Fc receptors on the
group A strain being studied.


62
The identification of two distinct Fc receptors from group A
strain 64/14/HRP was shown by probing four parallel nitrocellulose
blots containing the affinity purified type II Fc receptors with each
human IgG subclass. The 56,Q00 dalton protein (designated as type I la
Fc receptor) could bind human IgG subclasses 1, 2, and 4, whereas the
38,000 dalton protein (designated as type lib Fc receptor) only bound
human IgG subclass 3 (Fig. 3-5). Two other IgG3 myeloma proteins
were also tested and both reacted with the 38,000 dalton receptor (Fig.
3-5). Only one of the 1963 myeloma proteins tested was capable of
reacting with the type I Fc receptor and this immunoglobulin also
demonstrated a low level of reactivity with the 56,000 dalton (type
11 a) receptor (Fig. 3-6A). This suggests that the amino acid residues
which are important in the binding of the Fc region of human IgG},
I9G2, IgG4, and certain allotypes of IgG3, to the type I Fc
receptor, may also be part of the recognition site on the IgG molecule
for the type I la Fc receptor. In addition, the type I la Fc receptor
was capable of binding pig and rabbit IgG in contrast to the type lib
Fc receptor which could only bind human IgG3 (Fig. 3-10).
The studies on the effect of dipeptides on the binding of certain
human IgG subclasses to the Fc receptors on the surface of group A
strain 64/14/HRP provided further evidence for two distinct
functionally active forms of Fc receptor. Significant inhibition of
binding of IgG^, IgG2 and IgG4 to 64/14/HRP was observed with
glycyl-histidine and glycyl-tyrosine, while IgG3 binding was not
markedly effected (Fig. 3-3). Tyrosine and histidine are known to be
important residues in the binding of IgG to the type I Fc receptor


63
(Deisenshofer et_ aj_., 1978; Recht et^ al_., 1981; haake e^ a]_., 1982;
Shimizu et al_., 1983), but in these studies only the type I la receptor
was inhibited. No effects were observed on the interaction of IgG with
either the type I or type III Fc receptors (Fig. 3-4).
Although the type 1 la and type 11b Fc receptors are functionally
and physicochemically distinct, they were shown to be antigenically
related (Fig. 3-11). Monospecific antibodies prepared against the type
I la or type lib Fc receptors showed similar patterns of inhibition of
the binding of both 125j labeled human IgG and 1251 labeled
human IgG3 to the group A strain 64/14/HRP. Also, both Fc receptors
were recovered when the heat extract was affinity purified on a column
of immobilized anti-type lib Fc receptor, indicating that the similar
inhibition curves produced with either monospecific antibody could not
solely be attributed to steric hindrance of closely linked Fc receptors
on the bacterial surface. Based on the results of the inhibition study
using pig and rabbit IgG (Fig. 3-9), however, the type I la and type lib
Fc receptors are probably located close to each other on the cell
surface.
To date, there have been two other reports describing the
isolation of an Fc receptor from group A streptococci. Havlicek (1978)
described the isolation of an Fc receptor from Streptococcus pyogenes
which had a molecular weight of approximately 100,000 daltons and was
recovered from the bacteria following acid extraction. Grubb et al.
(1982) reported the isolation of a type II receptor from a group A
streptococci, type 15, following alkaline extraction. This receptor
could only be isolated to homogeneity in the presence of high
concentrations of protease inhibitors and had an apparent molecular


64
weight of 29,500. The results presented in Table 3-1 indicate that I
was unable to recover Fc receptor activity following either acid or
alkaline extraction of the group A strain used. These findings would
suggest that the Fc receptors on group A streptococci nay represent a
heterogeneous group of molecules which are anti genically related.
The group A streptococcal Fc receptors I have described are anti-
genically and physicochemically distinct from Fc receptors isolated
from other bacteria. Antibodies prepared against the type I staphylo
coccal or type III group C streptococcal Fc receptors failed to react
with the group A Fc receptor and all three receptors had different
molecular weights, see Figures 3-5 and 3-12.
The studies reported here suggest that at least two functionally
and physicochemically distinct Fc receptors are present on the surface
of certain group A streptococci. To date, using an immunoblotting
technique to study expression of Fc receptors on individual bacterial
colonies (Chapter Two) I have been unable to find a strain that
expressed only the IgG3 selective Fc receptor. The abilty to isolate
such a subclass selective receptor has a variety of important practical
applications for the isolation and quantitation of human IgG3. The
importance of such receptors in the pathogenesis of certain strepto
coccal infections is unclear. The interaction between bacterial
products and components of the host immune system may explain some of
the post infection sequelae associated with infection by certain group
A streptococci.


CHAPTER FOUR
DISTRIBUTION OF THE TYPE II Fc RECEPTORS ON NEPHRITOGENIC AND
NO I -N E PHR IT OG E NIC GROUP A STREPTOCOCCI
Introduction
The importance of Fc receptors in the course of bacterial
infections and post-infection sequelae is not clear. Bacterial Fc
receptors have been postulated as virulence factors (Ginsberg, 1972;
Schalen, 1982; Christensen et_ al_., 1977,1978,1981) and a correlation
has been reported between the virulence of certain group A streptococci
and their ability to bind the Fc portion of human IgG (Burova et al.,
1980). Post-streptococcal glonerulonephritis, a complication that
occasionally occurs following a group A streptococcal infection, is
believed to result from deposition of complement fixing immune
complexes in the glomeruli which initiate a complex series of reactions
that result in the development of renal lesions (Levinsky, 1981).
In the studies described in this chapter, a series of
nephritogenic and non-nephritogenic group A streptococci were screened
for Fc receptor expression. This study was designed to determine
whether Fc receptors are more frequently associated with group A
strains with nephritogenic M serotypes, than with those of M serotype,
not associated with nephritogenic potential.
In Chapter Two, I have described a sensitive blotting technique
that detects Fc receptor expression on bacterial surfaces. In this
chapter I have applied a modification of this technique to study the
65


66
interaction of human IyG or human IyG subclasses with group A
streptococci recovered from patients who either did, or did not develop
post-streptococcal glomerulonephritis.
Materials and Methods
Streptococcal Strains
Eighteen of the streptococcal strains used in this study viere
obtained from the Rockefeller University collection and were a gift
from Dr. Vincent Fischetti. Fifteen of these strains were isolated
from patients with post-streptococcal glomerulonephritis. The
characteristics of each of these strains have been reported in detail
by Villarreal et_ al_. (1979). Other group A streptococal strains were
obtained from Dr. Elia Ayoub at the University of Florida, College of
Medicine and were either throat or skin isolates.
Dot-Blotting Procedure
Dot blots were performed using the Bio-Rad bio-dot microfi 1 tration
apparatus and a modification of the Bio-Rad procedure. A piece of
nitrocellulose previously soaked in 25 mM tris, 192 mM glycine, pH 8.3
and 20% v/v methanol (wash buffer) was placed in the apparatus. Two
fold serial dilutions of the bacteria were pipetted into the wells.
The bacteria were diluted in the wash buffer, startiny with approxi
mately 1 x 108 bacteria. The concentration of organisms was
standardized by measuring the optical density at 550 nm. After washing
the bacteria in each well with the above buffer, the nitrocellulose was
removed and washed four times in veronal buffered saline (VBS), pH
7.35, containing 0.25% gelatin and 0.25% Tween-20. Each wash was
carried out for a period of 15 minutes using 250 ml of buffer. The


67
nitrocellulose was then probed for three hours in the washing buffer
containing 2 x 105 cpn/ml of the appropriate 125i labeled human
IgG or human IgG subclass. After probing, the nitrocellulose was
washed four times in 0.01 M EDTA, 1 M NaCl, 0.25% gelatin, and 0.25%
Tween-20 (15 minutes each wash) and allowed to air dry. All washing
and probing steps 'were performed at ambient temperature. The
nitrocellulose blots were autoradiographed by exposing to Kodak XAR-5
film with an intensifying screen for 3-5 days at -70C.
Iodination
Human IgG and human IgG subclases were iodinated as described in
Chapter Three.
Results
The distribution of Fc receptors on the surface of 35 strains of
group A streptococci was studied using a dot-blotting procedure. The
results of the experiment probing nephritogenic and non-nephritogenic
group A strains with 125j labeled human IgG are presented in Fig.
4-1 and the corresponding radioactive counts for the individual sample
wells are presented in Table 4-1. A comparison of the results in Fig.
4-1 with ttie counts in Table 4-1 indicate that the intensity of the
spot on the autoradiograph correlates closely with the number of
125i counts bound to the bacteria attached to the nitrocellulose.
Binding was shown to be dependent on the concentration of bacteria used
in the assay, and non-specific binding was found, under the
experimental conditions chosen, not to be significant (Table 4-1). A
similar relationship between the intensity of the spot on an
autoradiograph and the counts bound to the bacteria was observed with
all of the labeled probes tested.


Fig. 4-1. Binding of 125j human IgG to nephritogenic and
non-nephritogenic yroup A streptococci.
The indicated numbers of bacteria were dotted onto nitrocellulose
and probed with 125j human IgG as described in Materials and
Methods.
Panel A Panel B
Strain
M-type
Strain
M-type
1.
B920
49+
1.
646
49+
2.
B512
4
2.
647
49+
3.
11434
12+
3.
648
1
4.
A992*
18
4.
650
22
5.
A995
57+
5.
652
NT
6.
A207
2
6.
653
"787"
7.
A928
55+
7.
654
11
8.
B281
12+
8.
SHS1
NT
9.
A547
NT
9.
SHS4
12+
10.
B931*
2
10.
SHS7
NT
11.
B905
2
11.
SHS8
NT
12.
A374
12+
12.
SHS9
NT
13.
B438
18
- 13.
SHS10
NT
14.
0897*
12+
14.
SHS14
NT
15.
B923
12+
15.
SHS16
1
16.
B915
49
16.
SHS17
12+
17.
F2030
1
17.
SHS18
12+
18.
B515
NT
-
NT Non-typable
+ Nephritogenic M serotypes (Stollernan, 1971)
Panel A contains fifteen nephritogenic strains and three
non-nephritogenic strains (*). Autoradiography was at -70C for 3 days
with an intensifying screen.
Panel B contains seventeen non-nephritogenic strains. Autoradiography
was for 1 day at -70C with ari intensifying screen.


69
A
I X lo8
5 x I07
2.5 x I07
1.25 x I07
I x I08
5 x I07
2.5 I07
1.25 I07
34 56789 10 II 12
# 4)
* 9
*
9
13 14 15 16 17 18
B
7 8 9 10 II 12
1 X
I08



5 x
I07



2.5 x
I07
%

1.25 x
I07



1 x
I08

5 x
I07

2.5 x
I07

1.25 x
I07
*
13
14
15 16 17
*


TABLE 4-1
Binding of 125i Human IgG to Mephritogenic and Non-Nephritogenic Group A Streptococci
A
1
2
3
4
5
6
7
8
9
10
11
12
1 x 108
883
1013
516
713
597
600
1083
597
618
612
901
689
5 x 107
736
799
562
614
549
565
858
546
609
578
689
630
2.5 x 107
672
660
516
524
577
537
723
488
542
550
624
624
1.25 x 107
651
608
531
495
517
523
585
500
529
520
557
566
1 x 108
658
568
558
554
550
836
512
5 x 10 7
608
511
572
542
571
753
495
2.5 x 107
549
503
531
536
535
709
512
1.25 x 107
548
552
552
515
538
652
504
Con-
13
14
15
16
17
18
trol


TABLE 4-1 continued
B
1
2
3
4
5
6
7
8
9
10
11
12
1 x 108
1299
1349
1002
14421
1103
1638
935
20763
588
3747
595
899
5 x 107
1208
1299
880
7013
916
1244
925
21088
632
2789
642
1002
2.5 x 107
1000
991
743
10830
734
885
725
14264
623
2322
641
1242
1.25 x 107
1088
1231
840
7044
762
1042
820
15782
599
2257
613
969
1 x 108
711
3332
759
677
778
585
5 x 107
624
1474
766
680
603
602
2.5 x 107
732
720
683
1679
618
604
1.25 x 107
653
1092
676
652
610
547
Con-
13
14
15
16
17
trol
Each nitrocellulose blot was cut into sections that contained individual dots. Each section was counted
using an LKB autoyanma counter.
A CPM from the blot in Fig. 4-1A
B CPM from the blot in Fig. 4-IB


72
Since the results obtained in Chapter Three indicate the presence
of at least two distinct Fc receptors on group A strains, the
experiments described above using 125j labeled human IgG as probe
were repeated on the nephritogenic strains with each 125j labeled
human IgG subclass. The results are presented in Table 4-2. In
general, the nephritogenic strains which were able to bind the 125x
labeled human IgG probe, showed no particular preference for any one
subclass. One strain, A547, did bind IgG3 to a greater extent than
any other subclass, while another strain, A928, had opposite binding
characteristics. These two strains might be useful in isolating the
type lib and type 11a Fc receptors, respectively (see Chapter Three).
Piscussion
The role of streptococcal products in generating post
streptococcal glomerulonephritis is not well understood. In 1982,
Boyle proposed that the Fc receptors and the M protein were critical
components and proposed the following hypothesis which contains these
elements:
1) Nephritogenic strains of streptococci produce an Fc-reactive
protein.
2) This protein is released alone, or associated with other cell
wall constituents and forms complexes with normal IgG which
efficiently bind and activate complement.
3) These complexes of Fc-reactive protein and IgG are either not
cleared or inefficiently cleared by the reticuloendothelial
system and lodge in the kidney. The presence of the
anti-phagocytic M protein within such a complex could inhibit
its efficient clearance.


73
TABLE 4-2
Interaction of Nepiiritoyeriic and Non-nephritogenic
Group A Streptococci with Hunan IgG Subclasses
STRAIN
[1-TYPE
TOTAL
IgG
IgGi
igG2
igG3
IgC4
1.
B920
49
++++
+++
++
++++
++
2.
B512
4
++++
+++
+++
++
+++
3.
11434
12
+/-
-
+/-
-
+/-
4.
A992*
18
++
+
+/-
++
+
5.
A995
57
+
-
+
-
+
6.
A207
2
+
++
+
+/-
+
7.
A928
55
++++
+++
+++
+/-
+++
8.
B281
12
+/-
-
+/-
-
+/-
9.
A547
NT
++
-
-
++++
+/-
10.
B931*
2
+/-
-
-
-
-
11.
B905
2
++++
++
+
++++
+
12.
A374
12
++
+/-
-
+
-
13.
B438
18
+++
++
+/-
++
4-
14.
D897*
12
+/-
-
-
-
-
15.
B923
12
+
-
+/-
-
-
16.
B915
49
+/-
-
-
+
-
17.
F2030
1
+/-
-
-
-
+/-
18.
B515
NT
++++
+++
+++
++
+++
* = non-nephritis causing strains
NT = nontypable strain
- = no detectable binding
+ = binding detectable with 1 x 1()8 bacteria
++ = binding detectable with 5 x 107 bacteria
-H-+ = binding detectable with 2.5 x 107 bacteria
++++ = binding detectable with 1.25 x 10? bacteria


74
4) Once in the kidney, these complexes are trapped in the
vicinity of the basement membrane and tissue damage results
both through the action of complement itself and by other
components of the host's cellular immune system recruited by
chenotactic split products of complement (e.g., C5a).
This hypothesis is summarized in Fig. 4-2.
To test the first element of this hypothesis, a rapid dot-blotting
procedure was developed which enabled me to compare the Fc receptor
expression on nephritogenic vs. non-nephritogenic group A streptococcal
strains. Fifteen out of the eighteen nephritogenic strains tested
(73%) were positive for Fc receptor expression (Fig. 4-1A), whereas 65%
of the non-nephritogenic strains also had Fc receptors on their surface
(Fig. 4-1B). Since the nephritogenic strains tested were library
strains, the possibility exists that these strains have lost Fc
receptor expression during subculture (Kronvall, 1973a; Christensen and
Oxelius, 1974; Freimer et_ al_., 1979). Although the majority of
non-nephritogenic stains tested were fresh isolates, non-nephritogenic
library strains were also tested and 71% of these library strains still
maintained Fc receptor expression.
In Chapter Three, two distinct Fc receptors viere isolated from a
group A streptococcal strain. One Fc receptor was capable of binding
human IgG subclass 1, 2, and 4, while the other Fc receptor was
specific for human IgG subclass 3. Lewis et_ al_. (1970) have reported
that the immunoglobulin subclass composition of the glomerular deposits
in human renal diseases was selective and did not reflect the normal
serum concentration of these proteins. Patients who had granular
deposits of immunoglobulin, which suggest an inmune complex


75
CLEARED
BY RES
TRANSPORTED
TO KIDNEY
I
Fig. 4-2
Proposed mechanism of the pathogenesis of post-streptococcal
glomerulonephritis.


76
pathogenesis, tended to have selective deposits of immunoglobulin
composed of a single or dominant subclass, usually IgGg (Lewis et
al., 1970). In light of these studies, the nephritogenic group A
strains were tested for reactivity with each human IgG subclass (Table
4-2), however, none of the Fc receptor-positive nephritogenic strains
showed selective binding to any particular human IgG subclass.
Since Fc-reactive proteins were found on the majority of both
nephritogenic strains and non-nephritogenic strains, these results
indicate that nephritogenicity probably requires other factors in
addition to the ability to produce Fc-reactive proteins. One factor
which has been shown to be important to this pathogenic process is the
anti-phagocytic M protein (Jacks-Weis et al_., 1982). The presence of
the M protein in these complexes might prevent their clearance from the
circulation. Such uncleared complexes could lodge in the kidney,
activate complement, and initiate the pathogenic process that leads to
kidney destruction. By contrast, complexes lacking this protein, e.g.,
staphylococcal protein A IgG complexes, would be effectively cleared
from the circulation and consequently an absolute correlation between
Staphylococcus aureus infections and nephritis would not be expected.
Certain M types of group A streptococci are correlated with
nephritogenic potential (Stullermari, 1971), however, not all group A
streptococci with those nephritogenic serotypes cause glomerulo
nephritis. Also, post-streptococcal glomerulonephritis is not always
caused by a strain with a nephritogenic M serotype. In these studies,
only 53% of the strains isolated from patients with glomerulonephritis
had a nephritogenic M serotype. Of these strains, 63% had Fc receptors
on their surface. These results are similar to those obtained from the


77
strains isolated from patients without glomerulonephritis, in which 65%
of the strains have Fc receptors on their surface. Consequently, the
results of this study suggest that no absolute correlation exists
between Fc receptor expression and nephritogenicity. Although both the
M protein and Fc receptors have been proposed as virulence factors
(Ginsberg, 1972; Schalen, 1982; Christensen et 1977,1978,1981),
more studies need to be done in order to determine all the factors
required for post-streptococcal glomerulonephritis. Villarreal et al.
(1979) has reported the occurrence of an extracellular protein isolated
from patients with post-streptococcal glomerulonephritis, however, this
protein, like the type II Fc receptor and the M protein, was sometimes
produced by streptococci obtained from patients without the disease.
No biological properties of this extracellular protein could be
determined, but based on its reactivity with normal and specific rabbit
antiserum, its properties were distinct from those of the type II Fc
receptor (Ohkuni et al_., 1983).
The dot-blotting procedure described in this chapter can be
applied to the study of other bacterial diseases. In a recent study, I
have applied this technique to study the expression of a variety of
different receptors on clinical bacterial isolates recovered frail
patients suffering from endocarditis (Yarnall et _al_., 1985). Receptors
for collagen type I and type III, Clq, fibrinogen, fibronectin, and
human IgG were studied. The results of this study failed to identify a
coiimon surface receptor that could account for the ability of these
bacteria to colonize damaged heart tissue.
Since the early studies of Koch (1903), it has been necessary to
fulfill a certain number of criteria before it is possible to establish


78
a cause-effect relationship between any infectious agent and a disease
process. In carrying out the studies reported in this chapter, I was
aware that it would not be possible to evaluate critically the
hypothesis summarized in Figure 4-2. The results I have obtained would
indicate that the ir.imunoblotting technique I have developed can be used
as an efficient approach for more fully characterizing individual
strains of bacteria isolated from patients with a specific disease.
While this approach will never lead to elucidating a definitive
pathogenic mechanism, the information obtained from these studies is
helpful in determining if any correlation exists between a particular
receptor or surface protein and an infectious agent isolated from
individuals with a common disease state.


CHAPTER FIVE
CONCLUSION
Bacterial Fc receptors have been known for several years. Myhre
and Kronvall (1981) have characterized the functional activity of these
receptors on the bacterial surface, identifying five basic types. The
type I staphylococcal Fc receptor (Larigone, 1982a) and the type III
group C streptococcal Fc receptor (Reis et _al_., 1984c,1984d,1985;
Bjorck and Kronvall, 1984) have been purified and extensively
characterized. Little information is available on the other three
types of Fc receptors. A type II Fc receptor which is found on certain
strains of group A streptococci has been isolated (Grubb et a]_., 1982),
but with low yield, and the functional activities of the isolated type
II Fc receptor were not characterized.
In this study, I have described the isolation and characterization
of the type II Fc receptors from a mouse-passaged group A
streptococcus. A method was developed which enabled me to select an
individual Fc receptor-rich substrain from which to isolate the type II
Fc receptors. The type II Fc receptors were recovered in high yield
and were composed of two molecular weight forms that were anti geni cally
related, but functionally distinct. The 56,000 dalton receptor was
capable of binding human IgG subclasses 1,2 and 4, pig, and rabbit IgG.
The 38,000 dalton receptor could only bind the Fc region of human IgG
subclass 3. This is the first report of a unique receptor for a
particular subclass of human IgG.
79


80
The isolation of an Fc receptor which binds the Fc region of human
IgG3 has several practical applications. It can be used for
separating or depleting IgG3 from serum or secretions by immobilizing
the Fc receptor on sepharose. The IgG3~specific Fc receptor can be
radiolabeled or enzyme-linked for use in assays to detect and quantify
I9G3. This would be beneficial in diagnosing diseased states in
which the production of an IgG subclass is restricted to IgG3. For
example, Beck (1981) observed that antibodies directed against the
rubella virus were primarily of the IgG3 subclass. Antibodies
against thrombocytes in the serum of patients with idiopathic
thrombocytopenic purpura were demonstrated by Karpatkin et al_. (1973)
to be limited to IgG^. In addition, an extensive study by Natvig et
al. (1967) using Gn-specific antisera showed that the Rh antibodies in
the sera of mothers after an incompatible pregnancy belong to the
IgG3 subclass. The IgG3 Fc receptor might be useful in exploring
the mechanism of the IgG3 restriction in these diseases.
The role of group A streptococcal Fc receptors in the pathogenesis
of infection or post-infection sequelae is not clear. The distribution
of the Fc receptors on nephritogenic and non-nephritogenic group A
streptococci was studied, but no absolute correlation could be
established. The biological activities of the type II Fc receptors can
now be explored using the purified Fc receptors in both in vivo and in
vitro systems. Complement activation, mitoyenesis, and the nature of
Fc receptor-IgG complexes can be examined to clarify the role of Fc
receptors as virulence factors.
The methods described in this study to detect secreted and cell
associated Fc receptors, and to solubilize Fc receptors can be used in


81
future studies to explore new directions. First, streptococcal strains
can be treated with various mutagens or antibiotics to create a strain
which can secrete Fc receptors. Methici11 in has been used to isolate
strains of Staphylococcus aureus that produce only extracellular
protein A (Winblad and Ericson, 1973). An enhancement of the formation
of extracellular protein A was also achieved by growing Staphylococcus
aureus in the presence of puromycin (Movitz, 1976). These approaches
can be applied to streptococci to produce a mutant strain which
secretes Fc receptors.
With the discovery of a unique receptor for the Fc region of
IgGs, it is possible that different strains of streptococci have Fc
receptors that are specific for other human IgG subclasses. Using the
techniques described in Chapter Three, one can search for new unique Fc
receptors. Identification of Fc receptors for each human subclass
would be of great value in identifying and isolating IgG subclasses for
use in immunoanalytical and imrnu nodi agnostic assays and for studying
the fine structure of IgG constant domains.
Finally, the ability to screen large numbers of individual
bacterial colonies would be useful in identifying clones carrying a
specific gene that codes for surface receptors or proteins. Isolation
of a bacterial vector with the gene for a group A streptococcal Fc
receptor could be accomplished using this technique. Once the gene is
identified and cloned, several studies could be done to clarify the
loss of Fc expression observed on subculture. Also, specific mutations
of the cloned gene could determine if group A streptococcal Fc
receptors are virulence factors and whether or not they are involved in
the pathogenesis of post-infection sequelae.


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