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Effect of heat-killed Listeria monocytogenes and E. coli
01572H7 on function of human eosinophil and basophil-like
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presented by
Sridhar Samineni

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MS degree? Food Science

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2/05 p:/ClRC/DateDue.indd-p.1

EFFECT OF HEAT-KILLED LIS T ERIA MONOC Y T OGENES AND ESCHERICHIA
COLI OIS7IH7 ON FUNCTION OF HUMAN EOSINOPHIL-LIKE CELLS AND
BASOPHIL-LIKE CELLS

By

SRIDHAR SAMINENI

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science & Human nutrition

2005

ABSTRACT
EFFECT OF HEAT-KILLED LISTERIA MONOCYTOGENES AND ESCHERICHIA
COLI 0157:H7 ON FUNCTION OF HUMAN EOSINOPHIL-LIKE CELLS AND
BASOPHIL-LIKE CELLS
By

Sridhar Samineni

Using a human cell line model, we tested the hypothesis that exposure to heat-killed
Listeria monocytogenes (HKLM) and Escherichia coli 0157:H7.(HKEC) inhibits cell
proliferation, IL-4, IL-5, IL-13, eotaxin, ECP and EDN production, expression of CCR3
and FceRIa by the eosinophils and basophils. Whereas both pathogens inhibited
proliferation of eosinophil-like cells, only HKEC inhibited proliferation of basophil-like
cells. Both bacteria significantly increased the production of EDN by eosinophil-like
cells, and IL-5, IL-13 and eotaxin (CCLl 1) by basophil-like cells. Notably, both bacteria
inhibited IL-4 production by basophil-like cells and CCR3 expression on eosinophil-like
cells. Neither pathogens significantly altered the high affinity IgE receptor expression on
basophil-like cells. The purpose of this work was not to test what happens when allergic
humans are exposed to heat-killed bacteria. Rather our goal was to determine if these
bacteria can inhibit specific gene expression (such as IL-4, eotaxin and CCR3), so that
the active component of the organism could be isolated and used in future animal model
studies to asses protective therapeutic effects. Our findings that both bacteria specifically
inhibit IL-4 - a critical type—2 cytokine required for allergic responses, and CCR3 — a
major chemokine receptor implicated in allergy pathogenesis suggest the need for future

studies aimed to identify the IL-4 and CCR3 inhibiting components of these bacteria.

Dedicated to my parents, brother and all the people who helped me. . ..

iii

ACKNOWLEDGEMENTS

The work was accomplished with the help and support of numerous individuals. First I
must thank my major advisor, Dr. Venu Gangur for his guidance and continuous moral
and financial support throughout M.S program. He showed me different ways to
approach a research problem and the need to be persistent to accomplish any goal. I
would like to thank the members of my thesis committee, Dr. Elliot Ryser and Dr. Gale
Strasburg for their enthusiastic support and guidance. Besides my advisors, I would like
to thank Dr. Hanem mahamood (Post-Doc), Caleb Kelly, Dr. Norman Hord and Dr.
Fenton Jennifer for helping me with the protein array experiments and analysis of data
and Dr. Zhinong Yan for his assistance in preparation of heat-killed bacteria. I would like
to thank the rest of my colleagues (Lalitha Navuluri, Neil Birmingham, and Sitaram
Parvathaneni) for their help. I would like to thank Dr. Louis King and Dr. Pam Fraker for

assistance with flow cytometry experiments.

iv

TABLE OF CONTENTS

List of Tables ........................................................................
List of Figures ........................................................................
Abbreviations ........................................................................
Chapter 1: Introduction ...........................................................
Chapter 2: Review of literature ..................................................
Chapter 3: Materials & Methods ................................................
Chapter 4: Results ..................................................................
Chapter 5: Discussion .............................................................
List of References ...................................................................

Appendix ..............................................................................

Page
vi
vii

ix

19
27
54
61

70

Table 2.1

Table 2.2

Table 5.1

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

LIST OF TABLES

Comparison of primary human eosinophils and HL-6O clone 15
derived eosinophil-like cells ...............................................

Comparison of human basophils, mast cells and KU812 basophil-
like cells .......................................................................

CCR3 Antagonists: Relative specificity and potency ...................

Gene products that were affected by HKLM in human eosinophil-
like cells .......................................................................

Gene products that were affected by HKEC in human eosinophil-
like cells .......................................................................

Gene products that were unaffected by HKLM in human
eosinophil-like cells .........................................................

Gene products that were unaffected by HKEC in human eosinophil-
like cells .......................................................................

Gene products that were affected by HKLM in human basophil-like
cells ..........................................................................

Gene products that were affected by HKEC in human basophil-like
cells ..........................................................................

Gene products that were unaffected by HKLM in human basophil-
like cells .......................................................................

Gene products that were unaffected by HKEC in human basophil-
like cells .......................................................................

vi

14

16

58

7O

71

72

72

73

75

76

76

Figure 3.1

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

LIST OF FIGURES

Human cytokine/chemokine/ growth factor protein array
membrane. ...............................................................

Effect of HKLM on proliferation of human eosinophil-like

cells ...........................................................................

Effect of HKEC on proliferation of human eosinophil-like
cells ........................................................................

Effect of HKLM on proliferation of human basophil-like
cells .........................................................................

Effect of HKEC on proliferation of human basophil-like cells. . ..

Effect of HKLM on cytokine/chemokine production by human
eosinophil-like cells ...................................................

Effect of HKEC on cytokine/chemokine production by human
eosinophil-like cells ...................................................

Effect of HKLM on cytokine/chemokine production by human
basophil-like cells ......................................................

Effect of HKEC on cytokine/chemokine production by human
basophil-like cells ......................................................

ECP production by HL-6O clone 15 cells cultured with and
without 0.5 mM butyric acid ..........................................

EDN production by HL-6O clone 15 cells cultured with and
without 0.5 mM butyric acid ........................................

ECP production by human eosinophil-like cells cultured with and
without HKLM and HKEC. ....................................

EDN production by human eosinophil-like cells cultured with and
without HKLM and HKEC. ......................................

Basal level expression of CCR3 chemokine receptor by human
eosinophil-like cells ................................................

Effect of HKLM on CCR3 receptor expression by human
eosinophil-like cells ................................................

vii

24

28

29

3O

32

33

34

36

37

38

39

40

41

43

45

Figure 4.15

Figure 4.16

Figure 4.17

Figure 4.18

Effect of HKEC on CCR3 receptor expression by human
eosinophil-like cells ................................................

Basal level expression of FceRI receptor on human basophil-like
cells .....................................................................

Effect of HKLM on FceRI receptor expression by human
basophil-like cells ...................................................

Effect of HKEC on FceRI receptor expression by human basophil-
like cells ............................................................

viii

47

51

53

HKLM
HKEC
IL
MCP

MDC

NAP
RANTES
SDF
TARC
Th

OVA

lg

ECP
EDN

ELISA

LIST OF ABBREVIATIONS

Heat-killed Listeria monocytogenes
Heat-killed Escherichia coli 01 57:H7
Interleukin

Monocyte Chemoattractant Protein
Macrophage Derived Chemokine

Monokine Induced by interferon-y
Macrophage Inflammatory Protein

Neutrophil Activated Peptide

Regulated on Activation, Normally T-cell Expressed and Secreted
Stromal cell Derived Factor

Thymus and Activation-Regulated Chemokine
T helper lymphocyte

Ovalbumin

Immunoglobulin

Eosinophil Cationic Protein

Eosinophil Derived Neurotoxin

Enzyme Linked Immunosorbent Assay

ix

CHAPTER 1: INTRODUCTION

Allergic disorders such as asthma, allergic rhinitis (hay fever), food allergies, and atopic
dermatitis are significant public heath problems. Food allergies in particular ofien lead to
life threatening systemic anaphylaxis. Allergic reactions are immune mediated adverse
responses to common environmental antigens such as pollen, animal dander and food.
These disorders affect up to 20% of the population in industrialized countries (Trujillo
and Erb, 2003) and in the United States nearly 50 million people suffer from allergies

(CDC, 2003).

Mast cells, basophils and eosinophils play a key role as effector cells of allergic
inflammation. Furthermore, IgE antibody, IL-4, eotaxin, ECP, EDN, FceRI and CCR3

are major molecules involved in the pathogenesis.

The prevalence of allergies has been increasing since the early 1980s (Kim and Drake-
Lee, 2003; Sheikh and Strachan, 2004). The increase in allergic diseases has been
attributed in part to improvements in personal hygiene and reduced exposure to
pathogens, bacterial products, infections and increased exposure to allergens during early
life (Crane, 2002; Matricardi, 2004). This concept is often termed as ‘the hygiene

hypothesis’.

According to the hygiene hypothesis suggested by Strachan et a1. (1989), infections and
allergies are inversely related. In recent years there has been a growing interest in the
relationships among infections, pathogens and allergic diseases (Douwes et al., 2004;
Hadley et al., 2005; Matricardi et al., 2000; Mojtahedi and Ghaderi, 2005; Platts-Mills et
al., 2005). Several animal model studies showed that L. monocytogenes and E. coli (EC)
in particular may prevent allergies (Li et al., 2003a; Li et al., 2003b; Yeung et al., 1998).
Thus, two studies have assessed the effect of heat-killed L. monocytogenes (HKLM) on
food allergies in animal models (Frick etal., 2005; Li et al., 2003b). These studies
showed that HKLM inhibited the allergen specific IgE, airways eosinophilia and Th2
cytokine responses (Hansen et al., 2000; Li et al., 2003b). Notably these studies did not

examine the effect of L. monocytogenes on the function of basophils or eosinophils.

Li et al. (2003) showed that treatment with heat-killed E. coli (HKEC) can induce long
term downregulation of peanut induced allergenic immune responses in a mouse model.
In one other study using oral probiotic E. coli and allergic responses such as OVA-
specific IgE, total IgE, and IgG1 were suppressed in a mouse model (Kim etal., 2005).
Notably these studies did not examine the effect of E. coli on the function of basophils or

eosinophils.

Since basophils and eosinophils constitute only a small proportion of peripheral blood
leukocytes (O-5%), they are difficult to obtain in large numbers (Franklin, 2003).
Therefore, several studies have used the cell line KU812 as a model to study basophils

and HL-6O clone 15 derived cells as a model to study eosinophils.

Rationale and statement of the problem: Studies have been done looking at the effect
of HKLM and HKEC on allergic diseases in a mouse model. We are not aware of
previous studies examining the effect of HKLM or HKEC on basophil or eosinophil
function. Furthermore, there is little evidence for direct interaction between L.

monocytogenes or non-invasive E. coli OlS7:H7 and basophils and eosinophils.

This study was conducted with following aims and hypothesis

Aim 1: To test the hypothesis that HKLM and HKEC inhibit the proliferation of human

eosinophil-like cells and basophil-like cells.

Aim 2: To test the hypothesis that HKLM and HKEC inhibit the IL-4, IL-5, IL-13 and

eotaxin production by human eosinophil-like cells and basophil-like cells.

Aim 3: To test the hypothesis that HKLM and HKEC inhibit the ECP and EDN

production by human eosinophil-like cells.

Aim 4: To test the hypothesis that HKLM and HKEC inhibit the CCR3 receptor

expression by human eosinophil-like cells.

Aim 5: To test the hypothesis that HKLM and HKEC inhibit the high affinity IgE

receptor (Fce RIoz) expression by human basophil-like cells.

The purpose of this work was not to test what happens when allergic humans are exposed

to heat-killed bacteria. Rather our goal was to determine if these bacteria can inhibit

specific gene expression (such as IL-4, eotaxin and CCR3), so that the active component
of the organism could be isolated and for therapeutic effects assessed in future animal

model studies.

CHAPTER 2: REVIEW OF LITERATURE

This review of literature (i) summarizes the hygiene hypothesis for allergies; (ii) assess
the role of L. monocytogenes and E. coli (not 0157zH7) in allergic disorders as reported
in the literature and (iii) provides an overview on the biology of eosinophils and
basophils. It also compares and contrasts the biology of eosinophils, basophils and mast
cells with the human eosinophilic cell line (HL-60 clone 15 derived) and human

basophilic cell line (KU812) model systems used in this study.

Hygiene hypothesis:

In 1989, Strachan proposed the hygiene hypothesis to explain the increased prevalence of
allergic diseases in industrialized countries. According to this hypothesis, allergic
diseases may be prevented by exposure to infectious agents during early childhood.
Therefore, the rise in allergic diseases over the last three decades in developed countries
has been attributed in part to improved environmental conditions and reduced microbial

exposure.

Numerous reports suggested that hygienic environment in modernized societies may be
partly responsible for the increased prevalence of atopic diseases. Reductions in allergen
sensitization and atopic disease have been found in children of farmers, children with pets
and siblings in their homes, children raised in day-care, and children of large families

(Matricardi et al., 1998; Ring et al., 1999; Shaheen etal., 1996; Svanes et al., 1999).

Merchant et al (2002) investigated the relationship between asthma, allergies and farm
exposure. The aim of this epidemiological study was to estimate asthma prevalence and
assess whether farm exposures result in reduced atopy, allergic disease and asthma, while
taking into account multiple personal and other environmental risk factors, among this
cohort of farm children. In children, farming as the parental occupation was significantly
associated with lower rates of sneezing attacks, allergic sensitization, wheezing, itchy

skin rash and allergic rhinitis (Riedler et al., 2000, Von Ehrenstein et al., 2000).

Kramer et a1. (1999) suggested that early day care attendance (6—11 months) 6% children
from small families in Germany (up to three individuals) afforded 5 to 13 years of

protection against allergic sensitizations and hay fever. These findings were supported by
researchers in Arizona, US (Haby et al, 2000). Attendance at day care during the first six
months of life reduced the relative risk of asthma. Children exposed to other children had

more wheezing at 2 years of age, but less wheezing from age 6 to 13.

The intestinal microflora established during infancy may be a source for the induction of
immune deviation and the flora composition may determine whether allergy disorders
develop. Bjorksten et a1. (2001) investigated the relationship between allergies and
intestinal microflora. Their study found that children who developed allergies during the
first two years of life were less often colonized with enterococci, bifidobacteria, and
bacteroides, and had higher stool counts of Staphylococcus aureus and Clostridium

compared to healthy infants.

Kalliomaki et al. (2000) investigated the effect of probiotic bacteria on atopic eczema in a
double blind placebo control study. Results showed that frequency of atopic eczema in a
Lactobacillus GG-treated group was half of that in a placebo-treated group, suggesting
that Lactobacillus GG effectively prevented early atopic disease in high risk children.
Another study showed that Lactobacillus casei inhibited antigen-induced IgE production

by inducing interleukin-12 (IL-12) secretion by macrophages (Shida et al 1998).

Hessel et al. (2000) showed that gram-positive and gram-negative bacteria produced
different cytokines in human peripheral blood cells. The difference in OVA- specific
fecal IgA and serum IgGl levels among the experimental bacteria suggest different routes

for allergy inhibition.

Gereda et al. (2000) investigated the relationship between endotoxin exposure, type 1 T—
cell development, and allergen sensitization in infants at high risk of asthma. This study
showed that the homes of allergen-sensitized infants had significantly lower house dust
endotoxin levels than those of non-sensitized infants, and levels correlated with IFN-y—
producing T cells, but not with IL-4, IL-5, or IL-13 producing cell proportions. Exposure
to various microbial products early in life may explain the low prevalence of allergy
among children of farmers (Leynaert et al, 2001, Riedler et al 2001) and the reported

inverse relationship between levels of endotoxin house dust and prevalence of allergy.

In a murine model, oral bacterial extracts (comprised of lyophilized fractions of several
common respiratory tract bacterial pathogens) induced an increase in IFN-g and IgG2b,
and decrease in IgG1 and IL-4. Their results demonstrated that these bacteria] extracts

function by enhancing postnatal maturation of Th1 function (Bowman et al 2001).

Matricardi et al. (2000) investigated the relationship between foodbome, oro-fecal
exposure and atopy and allergic asthma in Italian cadets. Overall, respiratory allergies
and atopy were less frequent in people who were exposed to oro-fecal and foodbome
microbes such as Helicobacter pylori, T axoplasma gondii and hepatitis A virus, but not to

other microbes (such as mumps, measles, chicken pox) transmitted through other routes.

Several studies during the past eight years have provided controversial evidence on the
role of infections vs. allergy (Coheteta1., 2004; Hadley etal., 2005; Johnston and
Openshaw, 2001; Matricardi et al., 2000; Purvis et al., 2005; Radon et al., 2004). In the
following sections evidence on the role of L. monocytogenes and E. coli in allergy has
been reviewed.

L. monocytogenes exposure and allergy: Listeria monocytogenes, a Gram positive,
non-spore forming, motile, facultative intracellular organism causes listeriosis. It can be
isolated from soil, silage, and other environmental sources. Manifestations of listeriosis
include septicemia, meningitis, encephalitis, and intrauterine or cervical infections in

pregnant women, which may result in spontaneous abortion or stillbirth.

It has been shown that L. monocytogenes alleviates anaphylactic symptoms and reduces
allergen-specific IgE and Th2 cytokine (IL-4, IL-5, and IL-13) responses in murine
models. Frick et al. (2005) used a canine model to study the effect of HKL on peanut and
milk induced anaphylaxis. Their results suggest that vaccination of dogs allergic to
peanut and milk with HKL plus allergen, followed by oral challenge with peanut or milk,
HKL, and peanut markedly reduced the peanut induced anaphylactic symptoms and
allergen-specific IgE, and increased the oral tolerance for these allergens (Frick et al.,

2005).

Another study investigated the effect of HKL on the ovalbumin induced airway hyper-
reactivity model of asthma in a murine model. Their results showed that HKL
dramatically inhibited airway inflammation, eosinophilia and mucus production,
significantly reduced OVA-specific IgE and IL-4 production, and dramatically increased
Ag-specific IFN-g synthesis (Hansen et al., 2000). Similar study has done by Mizuki and
colleagues using L. monocytogenes infection on ovalbumin induced asthma in a murine
model. They found that L. monocytogenes infection suppresses OVA-induced allergic
responses like airway eosinophilia, total serum IgE, OVA-specific IgE and Th2 (IL-4, 5,

and 13) responses in spleen cell cultures (Mizuki et al., 2001).

One other study investigated the effect of HKL on primary and secondary Ag-specific
immune responses in a murine model. Mice immunized with Ag-keyhole limpet
hemocyanin (KLH) mixed with HKL showed increased production of Th1 cytokines and

KLH-specific IgGZa antibodies, and decreased IL-4 cytokine production and KLH-

specific IgE antibody. Thus, HKL may be clinically effective in vaccine therapies for

allergy and asthma (Yeung et al. 1998).

Heat-killed Listeria (HKL) mixed with recombinant peanut proteins as an adjuvant on
anaphylactic reactions in a mouse model for peanut allergy. HKL significantly reduced
peanut-induced anaphylactic symptoms, and inhibited the peanut specific IgE, plasma
histamine, Th2 (IL-4, IL-5 and IL-13) cytokine production in spleen cells. These results
show that immunotherapy with modified peanut proteins and HKL is effective for
treating peanut allergy in this model (Li ct al., 2003). All these studies demonstrated that
HKL may mediate immune deviation from a pathological Th2-dominated response
toward a protective immune response in asthma and allergic diseases. Notably these
works did not study the effect of L. monocytogenes on the function of eosinophils,

basophils or mast cells.

E. coli exposure and Allergies: Three studies have been looked at effect of E. coli on
allergic diseases. Oral administration of E. coli MC4100 culture to murine model of
allergy, before OVA-sensitization. E. coli inhibited total IgE production and markedly
reduced OVA-specific IgE levels. Additionally, E. coli treated mice had significantly
fewer of de-granulated mast cells. These results demonstrate that administration of E. coli
inhibited OVA-specific IgE synthesis and thereby reduced the intensity of allergy

symptoms (Kim et al., 2005).

10

 

Another study examined the effect of HKE producing engineered Ara hl, 2, and 3 on
peanut allergy in a mouse model. Thus, HKE-MP123 (heat-killed Escherichia coli
producing engineered Ara hl, 2, and 3) suppressed peanut induced anaphylaxis
symptoms, peanut-specific IgE, plasma histamine, and IL-4, IL-5, IL-10, IL-13 in
spleenocyte culture and increased IFN-y and TGF-Bl levels. HKE alone also had the
same beneficial effects. Treatment with HKE-MP123 can induce long-terrn

"downregulation" of peanut hypersensitivity in this allergy model (Li et al., 2003).

Oral administration of probiotic E. coli to children of allergic mothers and colonization
after birth influenced the levels of some cytokines (IL-4, IFN-g and TGF-beta) and also

reduced the clinical manifestations of allergy (Lodinova et al., 2003, 2004).

Thus, although some studies examined the effect of E. coli on T-helper cell function in
the mouse model, we are not aware of previous studies examining impact of E. coli in

general or E. coli 0157:H7 in particular on the fiinction of basophils or eosinophils.

Role of eosinophils, basophils and mast cells in allergy:

Mast cells, eosinophils and basophils are important immune cells in mediating allergic
disorders (Galli, 2000; Sampson, 2000). The inflammatory responses associated with
atopic disorders are characterized by the recruitment of eosinophils, basophils, and
lymphocytes to the site of inflammation (Gangur et al., 2003; Gangur and Oppenheim,
2000). Basophils and eosinophils are circulating granulocytes. They are of myeloid origin

and develop from pluripotcnt stem cells found in bone marrow. These cells are released

11

‘— u— : J.’_&_‘hi;m._'. _ . _ .._._~ ..

from the bone marrow as mature cells and have little capacity for further development
and proliferation in peripheral tissues (Murakami et al., 1969). Mediators produced by

these cells profoundly influence the orchestration of allergic inflammation.

Role of eosinophils in allergy:

Eosinophils, first observed by Wharton Jones in 1846, play an important role in allergic
diseases (Franklin, 2003). They vary in size from 12 to 17 um. Their nucleus is usually
bi-lobed and they are characterized by distinctive granules in the cytoplasm. They have a
short half-life of 8 to 18 h in circulation and then migrate from circulation to tissues

where the epithelial surfaces are exposed to the external environment (Franklin, 2003).

Eosinophils also have been implicated as primary effector cells in some specific
eosinophil mediated diseases such as allergic eosinophilic oesophagitis, allergic
eosinophilic gastroenteritis, allergic colitis and food protein—induced proctocolitis
(Rothenberg, 2004; Rothenberg etal., 2001; Sampson, 2004). Activated eosinophils
secrete a variety of proinflammatory or tissue-damaging substances, including granular
proteins and several chemokines, and cytokines. Allergic/inflammatory airway diseases
such as asthma are characterized by infiltration of eosinophils into lungs (Giembycz and

Lindsay, 1999).

Peripheral blood has relatively few eosinophils (05%). Therefore it is difficult to obtain

them in large numbers (Franklin, 2003). HL-60 clone 15-differentiated eosinophils

12

present a useful means to perform studies on eosinOphil function. HL—6O clone 15, when
treated with 0.5 mM butyric acid, can differentiate into cells closely resembling human
eosinophil-like cells (Fischkoff, 1988). These eosinophil-like cells express many
characteristics of primary eosinophils as summarized in Table 2.1. These cells also
express the chemokine receptor (CCR)—3 which is critical in trafficking of eosinophils to
sites of allergic reactions (Badewa etal., 2002; Michail and Abernathy, 2004; Tiffany et
al., 1998). Recent studies using gene knockout mice showed a key pathogenic role of
eosinophils and CCR3-eotaxin chemokine system in a mouse model of ovalbumin-
induced allergic gastritis. Thus, they demonstrated that mice genetically deficient in
eotaxin (a ligand for CCR3) were protected from eosinophil-mediated allergic gastritis

(Mishra and Rothenberg, 2003).

Upon activation, they release granular proteins such as major basic protein (MBP),
eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil derived
neurotoxin (EDN) (Gundel etal., 1991; Harrison et al., 1999; Rosenberg and
Domachowske, 2001). These cytotoxic proteins are believed to play a role in the
development of subacute and chronic symptoms of allergy (Venge, 2004). In addition,
eosinophils are also sources for proinflammatory cytokines such as IL-4, lL-S, IL-6, IL-8
and IL-10 (Bandeira-Melo and Weller, 2005; Nakajima etal., 1996) and chemokines
such as CCLS (RANTES), CCL3 (MIP-lor), CCL2 (MCP-l), CCL11 (eotaxin) and
CCL24 (eotaxin-2) (Bandeira-Melo and Weller, 2005; Chiba et al., 2005). Eosinophils

express various membrane receptors for adhesion, cytokine and chemokines. IL-5 is a

13

Table 2.1 Comparison of primary human eosinophils and HL-60 clone 15- derived

eosinophil-like cells (Franklin et al., 2003)

 

 

Characteristics Primary human HL-60 clone 15 derived
eosinophils eosinophils

Morphological

Maturity Mature Immature
Nucleus Bi-lobed Irregular
Granules in cytoplasm + +/-
Staining Acid dye eosin Acid dye eosin
Functional

Proliferation - +

Major mediators in + +
cytoplasmic granules (ECP,

EDN, EPO, MBP)

Cytokine released upon + NR
activation. IL-4, IL-5 and IL-

13

Chemokines released upon + +
activation, MCP, MIP-l
alpha, RANTES, eotaxin and
eotaxin-2

High affinity IgE receptor _ _
expression

Chemokine receptor + +
expression (CCR3)

 

NR : Not reported

14

Key cytokine in the regulation of eosinophilia and eosinophil activation in humans (Kay
et al, 2004). Human eosinophils express several chemokine receptors such as CCRl,
CCR3, CXCR3 and CXCR4. CCR3 plays a key role in recruitment of eosinophils to
allergic inflammation sites by binding to eotaxins, CCL 7 (MCP-3), CCL13 (MCP-4) and

CCL5 (RANTES) (Bandeira-Melo and Weller, 2005; Uguccioni et al., 1997).

In our studies clone 15 HL-6O human eosinophilic cells as the in vitro model along with
HL-6O clone 15 cells that expressed CCR3 chemokine receptor and granular proteins
such as ECP, EDN, and EPO similar to human primary eosinophils (Tiffany et al., 1998;

Tiffany et al., 1995).

Role of basophils/mast cells in allergy:

Basophils and mast cells play a key role in the development of immediate hypersensitive
reactions, because these are the only cells that bear high affinity receptors for IgE
antibody- i.e., the allergy-causing antibody (FccRIoz). Table 2.2 shows the similarities and

some differences between mast cells, basophils and the KU812 basophilic cell line.

Upon activation via F ccha receptor both mast cells and basophils can produce a similar
spectrum of proinflammatory mediators, as well as certain cytokines and chemokines.
Both of these cells share some phenotypic and functional properties, but they differ in
some important aspects such as natural history, location and mediator content. In contrast

to mast cells, basophils are circulating granulocytes that typically mature in the bone

15

Table 2.2: Comparison of human basophils, mast cells and KU812 basophil-like cells

(Franklin et a1, 2003, Blom et al 1992, Galli et al., 2000)

 

 

Characteristics Human basophils Human KU812
mast cells basophilic cell
line

Morphological
Location Circulation Tissue NA
Granules + + +
Maturity Mature Mature Immature
Staining Basic dyes Cationic dye Basic dye
Functional
Proliferation - + +
Major mediators in + +
cytoplasmic granules +
(histamine and tryptase)
Major lipid mediators + + - +
released upon activation
LTC4 and LTB4.
Cytokine released upon + +
activation. IL-4, IL-5, IL- +
13, TGF-Bl, IL-10, IL-3
and IL-6.
Chemokines released upon + +
activation, CCL-2,7,8, +
CCL3, CCLS

+ + +
High affinity IgE receptor
expression
Chemokine receptor + + NR
expression (CCR3)

 

NR-Not reported: NA- Not applicable
IL- Interleukin, LTC4- Leukotriene C4, LTB4- Leukotriene B4

16

marrow, circulate in the blood as mature cells, and can be recruited into sites of
inflammatory responses (Galli et al., 2005). Whereas mast cells can be long-lived and can
re-enter the cell cycle to proliferate locally, mature granulocytes such as eosinophils and

basophils do not (Galli, 2000; Galli and Hammel, 1994; Wedemeyer et al., 2000).

Basophils were first identified and described in 1879 by Paul Ehrlich (Galli and Hammel,
1994; Franklin, 2003). Mature basophils range in size from 5 to 8 pm, they have a lobed
nucleus with the chromatin in the nucleus condensed. Granular contents within the
cytoplasm include glycosaminoglycans, which give a metachromatic appearance after
staining. Basophils selectively infiltrate into the skin, nose, lung and other tissue sites by
expressing specific adhesion molecules and chemokine receptors (CCR3 in particular)
that enable their migration from the circulatory system into tissues (Bochner and
Sterbinsky, 1991; Wimazal et al., 1999). They express a high affinity IgE receptor
(FceRI) that plays an important role in cellular activation. FceRI is a tetrameric receptor
that consists of one or chain, one 3 chain and two 7 chains. The most important functional
characteristic of basophils is their ability to bind to IgE via the or subunit of high affinity
IgE receptor (FceRIoz) (Blank et al., 1989; Blank et al., 1991). Allergen cross linking of
IgE bound to Fce RIoz leads to basophil degranulation and the release of several
inflammatory mediators including histamine, proteases, cytokines, chemokines and
leukotrienes. Basophils account for all histamine released in circulation (lshizaka et al.,
1972) and are a significant source for Type 2 cytokines such as IL-4 and IL-l3, which are
now considered critical components in the pathogenesis of allergic diseases

(Devouassoux et al., 1999).

17

IL-4- is a critical Th2 cytokine responsible for important pro-inflammatory functions in
asthma and allergic diseases, including T helper cell type 2 lymphocyte differentiation,
induction of IgE production, up-regulation of IgE receptors, promotion of eosinophil
transmigration into the lungs and mucus hypersecretion. IL-4 is a key therapeutic target
in treatment of allergic diseases (Steinke, 2004). Basophils also produce several
chemokines such as CCL3 (MIP-loz), CCL2 (MCP-l) and CCLS (RANTES) and also
express CCR3 chemokine receptor (Iikura et al., 2004; Li et al., 1996; Uguccioni et al.,

1997).

Since basophils constitute only 1% of peripheral blood leukocytes, they are difficult to
obtain in large numbers (Franklin, 2003). Basophilic differentiation of KU812 cells
occurs by the serum starvation technique. Thus, cells grown in a serum-free medium
develop characteristics of human primary basophils and mast cells such as high affinity
IgE receptor expression and histamine production (Blom et al., 1992; Franklin, 2003;
Galli et al., 2005). Therefore, several studies have used the cell line KU812 model. This
model represents an immature basophil precursor and was originally obtained from a
patient with chronic myeloid leukemia. While this model is useful for studying basophil
biology (Blom et al., 1992). This basophil-like cell line must be viewed with caution
given fundamental differences in morphology and function compared with primary

human basophils.

18

CHAPTER 3: MATERIALS AND METHODS

HL—60 Clone 15 (Eosinohilic cell line): The HL-60 clone 15 cell line was obtained from
the American Type Culture Collection (ATCC, Manassas, VA) and grown in culture
media {86% (v/v) RPMI-164O containing 1.5 g/L sodium bicarbonate and 25Mm HEPES
(Sigma, St.Louis, M0), 2 mM L-glutamine (Gibco, Carlsbad, CA), 10% (v/v) fetal
bovine serum (Sigma), 100 units/mL penicillin, 100 ug/mL streptomycin and 0.25 pg/mL
amphotericin (Gibco); pH 7.6} at 37°C and 5% C02 (Isotemp C02 incubators, Fisher
scientific, Pittsburgh, PA) Cells were maintained at a starting concentration of 5 x 105
cells/mL. Every 3 days, cells were centrifuged (500 rpm, 370C, 5 min), and resuspended
in fresh culture media at 5 x 105 cells/mL. For experiments, cells were cultured in 0.5
mM sodium butyrate (Sigma) for 3 days and then washed with culture media and re-
suspended in fresh culture media containing 0.5 mM sodium butyrate (Sigma) with and

without HKLM and HKEC at indicated concentration.

KU812 Cell line (Basophilic cell line): The KU812 cell line was obtained from the
American Type Culture Collection (ATCC) and grown in culture media {86% (v/v)
RPMI-1640 containing 1.5 g/L sodium bicarbonate and 25mM HEPES (Sigma), 2mM L-
glutamine (Gibco), 10% (v/v) fetal bovine serum (Sigma), 100 units/mL penicillin, 100
pg/mL streptomycin and 0.25 ug/mL amphotericin (Gibco); pH 7.2} at 37°C and 5%
C02 (Isotemp C02 incubators, Fisher scientific) The cells were maintained at a starting
concentration of 5 x 105 cells/mL. Every 3 days cells were centrifuged (500 rpm, 370C, 5

min), and re-suspended in fresh culture media at 5 x 105 cells/mL. For experiments cells

19

were first centrifuged and washed with serum-free culture medium. Then the cells were

cultured in serum-free culture medium with and without HKLM and HKEC for 4 days.

Preparation and heat killing of L. monocytogenes and E. coli 0157:H7:

The following bacterial isolates were kindly provided by Dr. Elliot Ryser (Michigan State
University): (i) Listeria monocytogenes: ILSI #1 (Scott A, clinical strain, outbreak in
Mass,l983), ILSI# 35 (Strain collected from sliced turkey outbreak), ILSI #33 (outbreak
isolated from hotdogs); and (ii) E. coli 0157:H7: AR strain, AD305 strain and AD317

strain.

A loop full of each bacterial isolate from a frozen stock culture (-80°C in TSB-YE and
10% glycerol) was inoculated into 10 ml of trypticase soy broth yeast extract (TSB-YE)
medium (30 g of trypticase soy agar (BD biosciences, MD, USA), 6.0 g of yeast extract
(Difco laboratories, Detroit, MI), 1000 ml of distilled water and autoclaved at 121°C for
15 min) and incubated at 37°C for 24 h, after which a loop full of culture transferred to
40 mL of TSB-YE medium in sterile 50 mL tubes and incubated at 37°C for 24 h.
Cultures were harvested by centrifuged (7000 rpm for 15 minutes) and washed in 1%
(w/v) sterile peptone (BD biosciences) and re-suspended in 20 mL of 1% peptone.
Bacteria were enumerated after 48 h of inoculation at 37°C. The bacterial cultures were
heat-killed by incubating in 50 mL tubes at 72°C for 6 min in water bath and confirmed
by inoculating TSA-YE and mix plates. After enumeration, heat-killed bacterial solution
was centrifuged (7000 rpm for 15 min) to concentrate the bacteria and supernatant was

discarded. Then, bacterial cultures were re-suspended in 2 mL of RPMI-l640 medium

20

approximately at 1X10“/mL and stored at -80°C in 1.0 mL aliquots. Heat-killed bacteria

were added to the cell cultures at bacteria: cell ratios of 0: l , 100:1 and 1000:1.

Experimental approach: Eosinophil-like cells and basophil-like cells were cultured in 3
mL volumes in duplicates (6 well tissue culture plates, BD biosciences) with or without

HKLM and HKEC at bacteria: cell ratios of 0:1, 100:1 and 100021 at 37°C, 5% CO2.Cells
and cell culture supematants for controls and treatments were collected at days 1, 3 and 5

for eosinophil-like cells and days 1, 2, 3 and 4 for basophil-like cells.

Determination of cell proliferation and viability: After specified incubation periods,
cells were mixed thoroughly and 40 ul of cell suspension was transferred to a 0.6 mL
eppendorf tube. Forty pl of culture medium and 80 ul of 2% trypan blue (Sigma) were
added to the tube and enumerated by manual counting of cells in hemocytometer
(Hausser scientific, Horsham, PA) under 40 x magnification of microscope (Fisher
scientific). The number of cells in top two and bottom two rows of center big square were

counted in field and cell number were calculated.

Protein Array Analyses: Protein array kits (RayBio® Human Cytokine Antibody Array
VI, Raybiotech Inc, Norcross, GA), were used according to manufacturer instructions
except that 2 ml of cell cuture supemtant was used for each membrane. The procedure for
protein array analysis and location of cytokines/chemokines/ growth factors and positive,
negative, blank controls on the protein array membrane are shown in Figures 3.1
respectively. The protocol is described below: Briefly, 2 ml of blocking buffer was added
to membrane and incubated for 2h at room temperature. Thereafter membrane was

incubated for overnight at 4°C with sample (cell culture supematants) and followed by

21

incubation with a cocktail of biotin-labeled antibodies for 90 min at room temperature.
Then membrane was incubated with HRP-conjugated streptavidin at room temperature

for 2 hours. Finally chemiluminescence signal was detected by exposing to film.

Protein array images were scanned in grayscale at 9600 dpi using HP scanner (Hewlett-
Packard, Palo Alto, CA). Quantity One software (version 4.5.1 basic, BioRad, Hercules,
CA) was then used to analyze the scanned images. This software converted each circle on
the film into a numerical density value relative to the positive and negative controls on
the film. These numerical values were entered into an Excel based program (RayBio
analysis tool- human VI and 6.1) that performed normalization and background
subtraction. These numbers were used to calculate the fold change between each control

and treatment group.

ECP and EDN ELISA: ECP and EDN ELISA kits were obtained from MBL
International and samples were screened according to manufacturer’s instructions as

described below.

EDN ELISA: The EDN ELISA sandwich kit (MBL International, Wobum, MA) was
used to measure human EDN. One hundred uL of each cell culture supernatant and
standard were added to wells coated with anti-human EDN monoclonal antibody and then
incubated for 60 minutes at room temperature (20-25°C). After washing, peroxidase

conjugated anti-human EDN polyclonal antibody was added to the microwells and

22

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24

incubated for 60 minutes at room temperature. Then plates were washed, after which the
peroxidase substrate was added and incubate for 10 min. A stop solution (100 pL of 0.5
M sulfuric acid) added to wells to terminate the enzyme reaction and to stabilize the color
development. The optical density (CD) of each well was measured in dual mode of at
450-620 nm using the KC4 software program (Synergy HT, Multifunction Reader,
BioTek). The concentration of human EDN in samples was determined using standard

curves generated from recombinant human EDN.

ECP ELISA: The procedure for performing the ECP indirect sandwich ELISA (MBL
International) was similar to the EDN ELISA except for the coating antibody (anti-human

ECP), standard (ECP) and peroxidase conjugated anti-human ECP polyclonal antibody.

Flow cytometric analysis of FccRIa receptor expression on basophil-like cells: For
human basophil-like cells, cell surface expression of FceRIoz was assessed by flow
cytometry following instructions provided by the antibody supplier (eBioscience, San
Diego, CA). In brief, cells were incubated with affinity purified anti- human FccRIoc-
chain antibody CRA-l (lug/ 106) (eBioscience) for 60 min at 4°C. Then the cells were
washed twice in wash buffer (PBS, 0.5% BSA; pH 7.5) and exposed to the FITC-labeled
anti-mouse IgG (eBioscience) for 60 min at 4 ° C. Cells were washed twice in PBS and
subjected to flow cytometry. As a negative control, mouse subclass-matched polyclonal
IgG2b k antibody (eBioscience) was used to stain the cells. Results are represented as
mean fluorescence intensity. Flow cytometric analysis was done by Dr. Louis King (Dr.

Pam Praker’s lab, Department of Biochemistry, MSU).

25

Flow cytometric analysis of CCR3 receptor expression on eosinophil-like cells: For
human eosinophil-like cells, cell surface expression of CCR3 was assessed by flow
cytometry. In brief, cells were incubated with purified anti—human CCR3 reagent
(lug/million cells) (Biolegend, San Diego, CA) for 60 min at 4 ° C. Then the cells were
washed twice with buffer and incubated with biotin goat anti-mouse IgG (Biolegend) for
60 min at 4 ° C. Following another wash , the cells were incubated with PE labeled
strepatavidin (Biolegend) for 30 min at room temperature. To remove unreacted
streptavidin-PE, cells were washed twice and then, re-suspended in fixation buffer (PBS,
2% paraforrnaldehyde). As a negative control, mouse subclass-matched IgG2b antibody

was used for staining. Results are presented as mean fluorescence intensity.

Statistics: All proliferation, cytokine and chemokine data were analyzed using the
unpaired student t-test on duplicate samples at a significance level of p<0.05. Statistics
were performed using Analyse-It computer software program. All experiments were done

in duplicates and each experiment was replicated twice.

“Images in this thesis/(Lssertation are mesented in color.”

26

CHAPTER 4: RESULTS

Effect of HKLM and HKEC on proliferation of human eosinophil-and basophil-like
cells

In order to determine the effect of HKLM and HKEC on proliferation of human
eosinophil- and basophil-like cells, cells were cultured with and without HKLM or HKE
bacteria: cell ratios of 0: 1 , 100:1 and 100021. Human eosinophil-like cells grown without
HKLM continued to proliferate for up to 5 days. HKLM had no effect on proliferation of
human eosinophil-like cells at bacteria: cell ratio of 100:1 after 3 days. Although cell
numbers decreased at bacteria: cell ratio of 1000: 1, this decrease was not statistically
significant (Figure 4.1). HKLM significantly inhibited the cell proliferation at 1000:]

bacteria: cell on day 5 (unpaired students t-test p<0.05).

When compared to the untreated cells, HKEC had no effect on proliferation of human
eosinophil-like cells at 100:1 bacteria: cell ratio. HKEC inhibited cell proliferation at
1000:1 bacteria: cell ratio and this inhibition was statistically significant on day 5 (Figure

4.2) (unpaired students t-test p<0.05).

Culturing human basophil-like cells with HKLM for up to 4 days had no major effect on

cell proliferation (Figure 4.3).

We tested the effect of HKEC on proliferation of human basophil-like cells during 4

days. Cells cultured at 1:100 cell: bacteria ratio had no effect on proliferation.

27

 

1.50

 

 

 

 

A —9‘- 1:0
'{5‘ —0— 1:100 HKLM l
7, —A— 1:1000 HKLM
C
.9 i
E 1.00 ~ RE
fl
8 4 j T
o
.2 _L
- T T
H5 0.50
L-
o y .1.
-° *
E
3
z
0.00 ‘ ' L
1 3 5

Days of culture

Figure 4.1. Effect of HKLM on proliferation of human eosinophil-like cells. Cells were
cultured with or without HKLM at 0:1, 100:1 and 1000:1 bacteria: cell ratio. Data are

expressed as mean i SE of cell densities (* unpaired student t-test p<0.05).

28

 

1.50

 

 

A —9— 1:0
'§ —0— 1:100HKEC T l
B —t— 1:1000 HKEC
C
.9
“E 1.00 ~
.9
E
0
a)
.2
H5 0.50 r'
L- _1_
(D
.D
E
3
z
000 l l l

 

 

 

Days of culture

Figure 4.2. Effect of HKEC on proliferation of human eosinophil-like cells. Cells were
cultured with or without HKEC at 0:1, 100:1 and 1000:] bacteria: cell ratio. Data are

expressed as mean :t SE of cell densities (* unpaired student t-test p<0.05).

29

 

1.50

 

 

 

 

A —6— 1:0
-E' + 1:100 HKLM
B + 1:1000 HKLM
C
.9
E 1.00 — T
12 T
5 :l:
0
.J.

S T T
u— 0.50 _
o
c .L J—
(I)
.0
E
3
z

1 2 3 4

Days of culture

Figure 4.3 Effect of HKLM on proliferation of human basophil-like cells. Cells were
cultured with or without HKLM at 0:], 100:], 1000:] bacteria: cell ratio. Data are

expressed as mean :1: SE of cell densities counted in two different wells.

30

In contrast, basophil-like cells grown at a 121000 cell: bacteria ratio inhibited

proliferation on day 4 (Figure 4.4) (unpaired students t-test p<0.05).

In summary: (i) both HKLM and HKEC inhibited proliferation of human eosinophil-like
cells only at bacteria: cell ratio of 1000:]; (ii) HKLM had no major affect on proliferation
of human basophil-like cells; and (iii) HKEC inhibited proliferation of human basophil-

like cells only at bacteria: cell ratio of 1000:].

Effect of HKLM and HKEC on cytokine and chemokine production by human
eosinophil-like cells and basophil-like cells.

Day 3 cell culture supematants used from eosinophil-like cell experiments were screened
for cytokine/chemokine/growth factor production by protein array analysis. Figures 4.5
and 4.6 summarize the effect of HKLM and HKEC on allergy relevant cytokine and
chemokine production by eosinophil-like cells respectively. HKLM significantly
increased the production of important pro allergic chemokines such as CCL5 (RANTES),
CCL] (I-309), CCL8 (MCP-3) and CCL20 (MIP-3) by human eosinophil-like cells
(Figure 4.5). HKEC significantly increased the production of chemokines such as CCL5
(RANTES), CCL24 (Eotaxin-Z), CCL22 (MDC), CCL] (I-309), CCL8 (MCP-3) and

CCL20 (MIP-3) by human eosinophil-like cells (Figure 4.6).

Cell culture supematants of human basophil-like cell experiments (Day 2) were screened

the cytokine/ chemokine production by protein array analysis. HKLM clearly increased

31

 

 

 

 

 

1.20
A "9— 1:0
E + 1:1000 HKE
7, 100 ——A— 1:100HKE T
C
.9
E 0.80 —
9
8 0.60 ~
(I)
.2
H5 0.40 —
L
8 *
E 0.20 -
3
z
1 2 3 4

Days of culture

Figure 4.4 Effect of HKEC on proliferation of human basophil-like cells. Cells were
cultured with or without HKEC at 0:1, 100:] and 1000:] bacteria: cell ratio. Data are

expressed as mean i SE of cell densities (* unpaired student t-test p<0.05).

32

15

9
a)
E

m 3
.C
O

E -3
0
LL

-9

-15

 

***

 

I

E.-
w.
m

 

 

 

 

 

 

l l l l 1 1 l l l l l l
‘3' L0 00 ‘- ‘— V LO N 00 O N LL
_1, _', v; —J ‘- N _l _l _l N N O
_ _ o _r _l o Q Q _| _r
-—-' o o o o o o o o (’3
0 0 O O
Cytokines Chemokines

Figure 4.5 Summary of protein array analysis: Effect of HKLM on cytokine and

chemokine production by human eosinophil-like cells (* p< 0.05, * * *p<0.001 student t-

test).

33

 

 

 

 

 

 

 

 

15

9- ***
(D * *
‘=” 3— ** a?
(U
5 @m w
0
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VI'OC'JFFVLONOOONLL
_l_'le_ls-N.J_I_INNO
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—00000000
00 00
Cytokines Chemokines

Figure 4.6 Summary of protein array analysis: Effect of HKEC on cytokine /chemokine
production by human eosinophil-like cells (* p< 0.05, * * p<0.01, * * *p<0.001 student

t-test).

34

The production of cytokines and chemokines such as IL-5, IL-13, CCL5 (RANTES),
CCL] (I-309), CCL7 (MCP-3), CCL8 (MCP-3), CCL11 (Eotaxin) and CCL24 (Eotaxin-
2) and decreased IL-4 production (Figure 4.7). HKEC increased important
cytokines/chemokines such as IL-5, IL-13, CCL5 (RANT ES), CCL] (I-309), and CCL24
(Eotaxin-Z) and decreased IL—4, CCL8 (MCP-2) and CCL7 (MCP-3) (Figure 4.8).

Effect of HKLM and HKEC on ECP and EDN production by human eosinophil-like
cells.

Culture conditions were optimized for ECP and EDN production. HL-60 clone 15 cells
treated with 0.5mM butyric acid produced significantly higher levels of ECP and EDN
compared to the control (Figure 4.9 and 4.10). Standard curve were used for calculation
ECP and EDN levels. By using these standard curves, we calculated the ECP and EDN

concentration in samples.

To assess the effect of HKLM and HKEC on ECP and EDN production eosinophil-like
cells, were cultured at bacteria: cell ratio of 0:1, 100:] and 1000:] for five days and after
which the culture supematants were screened for ECP and EDN. As evident, ECP
production was generally increased by HKLM treatment, but it was not statistically
significant (Figure 4.11). In contrast, HKLM significantly increased the production of

EDN by human eosinophil-like cells, but not HKEC (Figure 4.12).

In summary, HKLM and HKEC had no significant effect on ECP production by human

eosinophil-like cells; and In contrast to HKEC, only HKLM significantly increased EDN

35

 

15

 

 

 

 

 

 

 

***
w
9~ ’3‘ *
*1:
a) **
3...
(D
c _ mm @
O
1.3.3-
0
LL
-Qr-
_15L11111111111
filflmFFVI-DNQONLL
_J_'I‘T_l‘-N_I_I_JNNQ
_.__IQ_I_IQQQ_I_ICD
—00000000
00 00
Cytokines Chemokines

Figure 4.7 Summary of protein array analysis: Effect of heat-killed L .monocytogenes on
cytokine /chemokine production by human basophil-like cells (* p<0.05, * * p<0.01, * *

*p<0. 001 student t-test)

36

 

15 ***

  
 
 

 
 

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02920.:

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2929

     

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SCF *

lL-4 *

lL-5 "
lL-13 "
CCL5 *
CCL7
CCL8 *

CCL24 *
CCL20 '—
CCL22 ’

CCL1 “
CCL11 ”

 

 

Cytokines Chemokines

Figure 4.8 Summary of protein array analysis: Effect of heat-killed E. coli 0157:H7 on
cytokine /chemokine production by human basophil-like cells (* p<0.05, * * p<0.01, * *

*p<0.001 student t-test)

37

 

20
*— o trol ,
----- ufync Acrd
3 16 —
g ..
C
C 12 _ ll
.9 /
H I
(D I
b I
C I,
o ,I
8 8 " ***,I
o
0
S
u.1 4 —
0

 

 

 

 

Days of culture

Figure 4.9. ECP concentrations in HL-60 clone 15 cell supematants cultured with and
without 0.5 mM butyric acid. Data are shown as mean +/- SE (unpaired student t-test **

p< 0.01, *** p<0.001).

38

 

 

15
_ Control

A “““ 0.5 mM butyric acid
E 12 —

\

C)

5

.5 9 —

H

(D

b

8

o 6 r

c

O

0

Z

O 3 —

LU

0 ‘ 1 1 I

 

 

 

Days of culture

Figure 4.10. EDN concentration in HL-60 clone 15 cell supematants cultured with and
without 0.5 mM butyric acid. Data are shown as mean +/- SE (unpaired student t-test

p<0.05).

39

 

 
 
 

88888

 

 

  

 

 

85 250 :2: mmmmmmmm

Figure 4.11. ECP production by h an eosrnophrl like cells cu] ed With and without
HKLM and HKEC. X-axis shows bacteria cell ratios Data are sho as mean +/ SE.

 

 

 
 

 

 

 

85 260 :2: ........

Figure 4.12 EDN production by h an eosrnophrl like cells cu] e
HKLM and HKEC. X-axis shows bacteria: cell ratios Data are sh
unpaired student t-test p<0 05)

production by human eosinophil-like cells.
Effect of HKLM and HKEC on chemokine receptor CCR3 expression by human
eosinophil-like cells.

To determine the effect of HKLM and HKEC on chemokine receptor CCR3 expression

by human eosinophil-like cells, human eosinophil-like cells were cultured with heat-
killed bacteria at bacteria: cell ratios of 0:], 100:1 and 1000:] for 3 days. The cell surface
expression of CCR3 was measured by flow cytometry using anti-human CCR3 antibody.
Figure 4.13 shows basal level expression of CCR3 chemokine receptor on human

eosinophil-like cells.

Using this system, we tested the effect of HKLM and HKEC on CCR3 chemokine
receptor expression by human eosinophil-like cells. Both bacteria tested inhibited CCR3
expression on eosinophil—like cells by ~ 50%. This inhibition was clearly seen at bacteria:
cell ratios of 100:] and 100021 for HKLM. Whereas HKEC inhibition was only seen at

bacteria: cell ratio of 100:1 (Figures 4.14- 4.15).

Effect of HKLM and HKEC on chRI expression by human basophil-like cells.

To assess the effect of heat—killed bacteria on FceRI expression on human basophil-like
cells were cultured with HKLM and HKEC at bacteria: cell ratios of 0:1, 100:] and
1000:] for 48 h under serum-free conditions. Cell surface expression of chR1 was

measured by flow cytometry using the anti-FceRIoc chain antibody CRA-l.

42

 

100

3 -

El - -
p 75'; '-
II

‘5 501

1.. -

3 '.

E 25:

3 ..

z .

 

 

 

 

'UTU

o 1 2 3
10 10 10 10

Mean fluorescence intensity

Figure 4.13. CCR3 receptor expression on eosinophil-like cells. Cells stained with control

antibody (black line) or anti-CCR3 antibody (redline) were subjected to flow cytometry.

43

Figure 4.14 (A-B). CCR3 receptor expression by eosinophil-like cells cultured with
HKLM at (A) bacteria: cell ratio of 100:] (B) bacteria: cell ratio of 1000:] for 3 days.
HKLM treated cells stained with anti-CCR3 antibody (blue line) and control cells stained

with control antibody (black line) or anti-CCR3 antibody (red line).

44

Number of events

Number of events

 

1oo_

   
   
 
  

75{
50{

25{

 

 

o 1 2 a
10 10 10 10

Mean fluorescence intensity

 

 

1on-
75§
50{

25-_'

 

 

 

0 I '1 'W2 3
'10 '10 10 '10

Mean fluorescence intensity

45

Figure 4.15 (A-B). CCR3 receptor expression by eosinophil-like cells cultured with
HKEC at (A) bacteria: cell ratio of 100:] (B) bacteria: cell ratio of 1000:] for 3 days.
HKEC treated cells stained with anti-CCR3 antibody (blue line) and control cells stained

with control antibody (black line) or anti-CCR3 antibody (red line).

46

Number of events

Number of events

 

100.

U] ‘11
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47

Figure 4.16 shows the basal level expression of F ccRI on basophil-like cells cultured

without any bacteria.

Using this system, we tested the effect of HKLM and HKEC on FccRI receptor

expression by human basophil-like cells. As an overall trend, neither of these bacterial

pathogens had significantly affected FccRIa receptor expression by human basophil-like

cells (Figures. 4.17- 4.18).

“Images in this thesis/dissertation are presented in color.”

48

 

100

 

 

 

 

3 .

5 .

> 75':

111 - l

‘5 50- i

1.. -

g i

E 25-_

3 u’

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'10 10 10 10

Mean fluorescence intensity

Figure 4.16. FceRI receptor expression on basophil-like cells. Cells stained with control

antibody (black line) or anti-CCR3 antibody (redline) were subjected to flow cytometry.

49

Figure 4.17 (A-B). FccRI expression by basophil-like cells cultured with HKLM at (A)
bacteria: cell ratio of 100:1 (B) bacteria: cell ratio of 1000:] for 2 days.. HKLM treated
cells stained with anti- chRI antibody (blue line) and control cells stained with control

antibody (black line) or anti- chRI antibody (red line).

50

Number of events

Number of events

 

100.

 

o
10

 

 

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75{
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51

Figure 4.18 (A-B). FceRI expression by basophil-like cells cultured with HKEC at (A)
bacteria: cell ratio of 100:1 (B) bacteria: cell ratio of 1000:1 for 2 days. HKEC treated
cells stained with anti- chRI antibody (blue line) and control cells stained with control

antibody (black line) or anti- FceRI antibody (red line).

52

Number of events

Number of events

 

100.
75-}
50{

25{

 

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0
10

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10 10 '10

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53

CHAPTER 5: DISCUSSION

This study was undertaken to determine whether heat-killed L. monocytogenes (HKLM)
and E. coli 0157:H7 (HKEC) contain any components that have the capacity to inhibit
the production of allergy-relevant cytokines (such as IL-4, IL-5 and IL—l3), chemokines
(such as eotaxins), and cell surface expression of chemokine receptor (CCR3) and IgE
antibody receptor. The rationale was that, if they were to exhibit such activity then future
studies could be designed to isolate the active component of these bacteria and which
could be used for potential therapeutic purposes in animal models. Based on our findings,
evidence was obtained for the presence of IL-4 and CCR3-inhibiting components in both

HKLM and HKEC, but not other targeted genes.

Basophils and mast cells are the primary cell types that express the high affinity receptor
for IgE antibody; activation of this receptor is the initiator of an allergic reaction leading
to clinical symptoms of allergy (Fujimura et al., 2002; Mac glashan, 2005). The mast cell
model that we had obtained did not express a functional high affinity receptor for IgE.
Basophils can also produce the key allergy relevant cytokines such as IL-4, IL-5, IL-13
and chemokines (such as eotaxins) (Falcone et al., 2000; Galli, 2000; Falcone, 2003).
Consequently, we used a human basophilic cell line as a model system to asses the

impact of HKLM and HKEC on these gene products of importance in allergy.

Eosinophils express the chemokine receptor CCR3---a major receptor shown to be

required for allergic inflammation. CCR3 gene deficient mice exhibit reduced allergic

54

responses (Ma et al., 2002; Mishra and Rothenberg, 2003) and CCR3 is one of the
currently targeted chemokine receptors for novel drug development for allergy and
asthma (Gangur et al., 2003). Eosinophils are the primary producers of ECP and EDN---
key mediators of nerve damage and inflammation associated with eosinophil mediated
allergies including subset of food allergy (Hogan et al., 2001; Romano et al., 1997;
Rothenberg et al., 2001a). In view of these facts, we used a eosinophilic cell line model to
study the impact of HKLM and HKEC on all these gene products of importance in

allergy.

Our goal was not to study the impact of HKLM and HKEC on human allergic diseases,
because the cell line model that we used could not have been used for studying disease.
Furthermore, others had already studied the effect of these two bacteria on allergic
disease in mouse models, although they used E. coli strains other than 0157:H7 (Kim et
al., 2005; Li et al., 2003 a). Thus, we used these previous studies as a rationale to focus on
the two bacteria that we selected. Listeria monocytogenes served as a model Gram-

positive bacteria and E. coli 0157:H7 served as a model Gram negative bacteria.

Our results indicate that both bacterial strains inhibited the expression of IL-4 in
basophil-like cells and CCR3 in eosinophil-like cells. Earlier studies using mouse models
have shown that HKLM and HKEC inhibit T helper cell production of IL-4 (Hansen et
al., 2000; Li et al., 2003a; Li et al., 2003b; Yeung et al., 1998). However, they had not
studied IL-4 production by basophils or CCR3 expression. We also found that HKEC

inhibited production of MCP-2 and MCP-3 in basophils. Production of these two

55

chemokines is elevated in allergic diseases (Freyne et al., 2005; Fulkerson et al., 2004;
Shang et al., 2002). However, currently there is no evidence implicating an essential role
for these two chemokines—as opposed to CCR3 and IL-4, in allergy. It is possible that
they may contribute to allergic inflammation as simply secondary participants facilitating
additional inflammatory cell recruitment (Gangur and Oppenheim, 2000). Thus, it is

unclear if inhibiting MCP-2 and MCP-3 might be of therapeutic value in allergy.

It is notable that inhibition of IL-4 is of special importance, because IL-4 is the prototypic
Type-2 cytokine necessary for T helper-2 (Th2) responses. Furthermore, the allergy
causing IgE antibody production is Th2 dependent (Lange etal., 1998; Steinke, 2004).
Our data show that HKLM and HKEC inhibited IL-4 production by human basophil-like
cells. Earlier studies showed that both bacterial strains inhibited IL-4 production by
mouse T helper cells (Li et al., 2003a; Yeung etal., 1998). IL-4 can also be produced by
certain types of NK cells in mice (Yoshimoto et al., 1995). However, whether these
bacteria can also inhibit IL-4 production by NK cells remains to be tested. If HKLM and
HKEC inhibit IL-4 production by these three sources of IL—4, then they would be
expected to inhibit IL-4 dependent allergic responses and perhaps allergic disease as well.
IL-4 gene knockout mice with reduced allergic responses (Lange et al., 1998) and IL-4
have been targeted by biotech industries for developing novel anti-allergy drugs (Steinke,

2004)

We found that co-culture of HKEC and HKLM inhibited the expression of CCR3 on

eosinophil-like cells by about ~50%. CCR3 is the chemokine receptor for chemokine

56

ligands such as eotaxin, eotaxin-2 and eotaxin-3 (De Lucca et al., 2005; Gangur et al.,
2003). It was reported that CCR3 gene knockout mice are significantly protected from
allergic response (Ma et al., 2002). Inhibition of CCR3 in vivo using specific antibodies
or chemical antagonists has shown promising results in blocking allergic reactions in
mice and in humans (Gangur et al., 2003; Gangur and Oppenheim, 2000). Consequently,
extensive efforts have been undertaken by the biotech and the pharmaceutical industries
to develop selective CCR3 antagonists for potential use in allergic disorders (De Lucca et
al., 2005; Elsner et al., 2004). Thus, several types of products are being developed as
summarized in Table 5.1 (Gangur et al., 2003). Using a novel approach ---i.e., use of
human eosinophil-like cells and foodbome pathogenic bacteria, we have found evidence

for inhibition of CCR3 expression.

Our results showed that neither HKLM nor HKEC affected on the expression of FceR-I —
the high affinity IgE receptor. Although mouse model studies assessed the impact of
HKLM and HKEC on allergic response, they did not studied the expression of high

affinity IgE receptor (Hansen et al., 2000; Li et al., 2003a; Li et al., 2003b).

We also found that both HKLM and HKEC inhibited proliferation of eosinophil-like cells
and basophil-like cells in vitro. The in vivo relevance of these findings is unclear.
Previous studies have shown that mature human eosinophils and basophils lack the
proliferating capacity (Galli et al., 2005). Our model system represents only the immature
form of these leukocytes. We performed the proliferation experiments to determine the

dose vs. culture time points that would have minimum impact on cell viability so that we

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58

would have sufficient cell numbers for future experiments. Accordingly, experiments
described in aims 2 though 4 were performed on day 3 of culture when cell viability had

not been seriously impaired.

In this study we tested five hypotheses, among these hypothesis 1 and 4 were tested
positive and others were rejected. Our results showed that both bacterial strains
significantly increased production of several cytokines/chemokines and EDN production
in the model system. Does this mean that heat-killed bacteria, if consumed by humans
can aggravate allergic inflammation? Our goal was not to study allergic reaction per se
and the model system that we used does not have the power to address this important
question. F urtherrnore, previous animal model studies report inhibition of allergic

responses by HKLM and HKEC.

In summary, our data suggest that HKLM and HKEC contain components that inhibit IL-
4 production and CCR3 expression in human basophil-like cells and eosinophil—like cells,
respectively. Identification of the active components of these bacteria and further testing

in other cell types and animal models would reveal whether they may be suitable as novel

therapeutic agents in allergic diseases.

59

Future directions

(A). Identification of the active components of these bacteria and further testing in other
cell types and animal models would reveal whether they may serve as novel therapeutic

agents for potential use in allergic diseases.

(B). In this study only CCR3 and FceR-I receptor expression was studied. Future studies
aimed at studying the function of these receptors would be valuable. Some studies
suggested for future work include: (i) To study the chemotaxis of eosinophili like cells in
response to CCR3 agonists such as eotaxins; (ii) To study the calcium signaling events in
response to CCR3 agonists; these studies are especially important because inhibition of
CCR3 expression was only by ~50% in our study; and (iii) to study the release of

histamine by IgE receptor activation for basophil like cells.

60

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Appendix

Table 1 Other gene products that were affected by HKLM in human eosinophil-like cells

 

 

Gene product Significant alteration * Fold change“ P value***
Chemokines
CCL15 (MIP-l t 1.93 <0.01
delta)
Growth factors
Light 1 2.81 <0.05
PDGF-BBZ t 8.3 <0.001

 

*Significant alteration — Significant increase or decrease in cytokine/chemokine/ growth
factors production by HKLM treated as compared to untreated cells.

** Calculated by dividing specific cytokines/chemokines/growth factors production by
the cells that were treated with and without bacteria.

*** Significant differences from controls using the unpaired student t-test.

7O

Table 2 Other gene products of eosinophil-like cells that were affected by HKEC

 

 

Gene product Significant alteration* Fold change** P value***
Cytokines
TNF-a t 3.62 <0.05
TNF-[i t 2.26 <0.05
IL-2 1 3.49 <0.05
IL-l ra t 1.42 <0.05
Chemokines
CCL3 (MIP-l a) t 7.79 <0.001
CXCL9 (MIG) t 2.04 <0.05
Growth factors
PDGF-BBZ t 6.34 <0.05
GM-SCF t 4.5 <0.05

 

*Significant alteration — Significant increase or decrease in cytokine/chemokine/ growth

factor production by HKEC treated as compared to untreated cells.

** Calculated by dividing specific cytokines/chemokines/growth factors production by

the cells that were treated with and without bacteria.

*** Significant differences from controls using the unpaired student t-test.

71

Table 3 Other gene products of eosinophil-like cells that were unaffected by HKLM

 

 

Cytokines Chemokines Growth factors

IL—l ra, 1L-1aIL-16 CCL2(MCP-1) Angiogenin, BDNF,

1L-3, IL-6, IL-7, IL-10 CCL14(MCP-4) BLC, BMP-4, BMP-6

IFN-"y, TNF- a, TNF- 6 CXCL7(NAP-2) CK- 6 8.1, CNTF

1L-15, 1L-2, CCL3(MIP-1a) EGF, Fractalkine

TGF- 6 1, TGF- 6 3 CXCL8(MIG) GCP-2, GDNF
CCL17(TARC) IGFBP-l, 2, 3, 4
CCL-26(Eotaxin-3) IGF-l, PARC
CXCL12(SDF-l) Leptin, GM-SCF

M-SCF

Fit-3 ligand, NT-3

Table 4 Other gene products of eosinophil-like cells that were unaffected by HKEC

 

 

Cytokines Chemokines Growth factors
IL—la IL- 16 CCL2(MCP-1) Angiogenin, BDNF,
IL-3, IL-6, IL-10 CCL14(MCP-4) BLC, BMP-4, BMP-6
IFN-y CXCL7(NAP-2) CK- 6 8.1, CNTF
IL-lS, CCL17(TARC) EGF, Fractalkine
TGF- 6 1, TGF- 6 3 CCL-26(Eotaxin-3) GCP-2, GDNF
CXCL12(SDF-1) IGFBP-l, 2, 3, 4

IGF-l, PARC

Leptin

M-SCF,

Fit-3 ligand, NT-3

72

Table 5 Other genngroducts that were affected by HKLM in human basophil-like cells

 

 

Gene product Significant alteration * Fold change“ P value***
Cytokines
TNF-6 t 2.8 <0.05
IL-lO t 3.73 <0.01
IL-lS t 1.92 <0.01
IL-1 or t 1.62 <0.05
IL-1 6 t 2.41 <0.05
IL-6 t 4.42 <0.001
IL-3 1 6.76 <0.01
Chemokines
CXCL9(MIG) t 1.58 <0.05
CCL15 l 2.5 <0.05
(MIP-l 6)
Growth factors
NT-3 ‘1 1.83 <0.05
PARC t 2.74 <0.05
SDF-l t 2.55 <0.01
FGF—7 l 2.5 <0.05
BMP-4 1 5.61 <0.05
Fit-3-ligand 1 1.58 <0.05
IGF-l t 4.6 <0.05
Light t 2.92 <0.05

*Significant alteration — Significant increase or decrease in cytokine/chemokine/ growth

factor production by HKLM treated as compared to untreated cells.

73

** Calculated by dividing specific cytokines/chemokines/ growth factors production by
the cells that were treated with and without bacteria.

*** Significant differences from controls using the unpaired student t-test.

74

Table 6 Other gene products of basophil-like cells that were affected by HKEC

 

 

Gene product Significant alteration * Fold change“ P value***
Cytokines
IL-6 1‘ 4.22 <0.001
Chemokines
CXCL7(NAP-2) 1 1.64 <0.05
Growth factors
BMP-4 1 2.7 <0.05
IGF-l ‘1 4.45 <0.05
Light t 2.04 <0.05
NT-3 1 2.34 <0.05
FGF-7 1 3.48 <0.05
SDF-l 1 2.43 <0.01

 

*Significant alteration — Significant increase or decrease in cytokine/chemokine/growth

factors production by HKEC treated as compared to untreated cells.

** Calculated by dividing specific cytokines/chemokines/growth factors production by

the cells that were treated with and without bacteria.

*** Significant differences from controls using the unpaired student t-test.

75

Table 7 Other gene products of basophil-like cells that were unaffected by HKLM

 

 

Cytokines Chemokines Growth factors

IL-l ra, 1L-7 CCL2(MCP-1) BDNF

IFN-y, TNF- a, CCL14(MCP-4) BLC, BMP-6

TGF- 6 l, TGF- 6 3 CXCL7(NAP-2) CK- 6 8.1, CNTF

1L-2, IL-16 CCL17(TARC) Fractalkine
CCL-26 (Eotaxin-3) GCP-2, GDNF

IGFBP-l, 2, 3, 4
Leptin, GM-SCF
M-SCF

Table 8 Other gene products of basophil-like cells that were unaffected by HKEC

 

 

Cytokines Chemokines Growth factors
IL-l ra, IL-la IL-16 CCL2(MCP-1) Angiogenin, BDNF,
IL-3, IL-7, IL-10 CCL14(MCP-4) BLC, BMP-6
IFN-y, TNF- a, TNF- 6 CXCL8(MIG) CK- 6 8.1, CNTF
IL-15, IL-16 CCL17(TARC) EGF, Fractalkine
TGF- 6 1, TGF- 6 3 CCL-26(Eotaxin-3) GCP-2, GDNF
1L-2 CCL3(MIP-1a) IGFBP-l, 2, 3, 4
PARC
Leptin, GM-SCF
M-SCF,

Fit-3 ligand, NT-3

76