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Studies of lacrimal gland immune cell homeostasis in polymeric immunoglobulin receptor (pIgR) knockout mice and evaluation of monoclonal dimeric IgA producing rabbit hybridomas

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Studies of lacrimal gland immune cell homeostasis in polymeric immunoglobulin receptor (pIgR) knockout mice and evaluation of monoclonal dimeric IgA producing rabbit hybridomas

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STUDIES OF LACRIMAL GLAND IMMUNE CELL HOMEOSTASIS IN
POLYMERIC IMMUNOGLOBULIN RECEPTOR (pIgR) KNOCKOUT MICE AND
EVALUATION OF MONOCLONAL DIMERIC IgA PRODUCING RABBIT
HYBRIDOMAS

by
Mubashera Kothawala

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2010

Copyright 2010 Mubashera Kothawala

ii
ACKNOWLEDGEMENTS

I thank my guide and thesis advisor, Dr. Sarah Hamm-Alvarez for her guidance
and especially her enthusiasm and detailed discussions which helped mold this project. I
thank my committee members, Dr. Okamoto and Dr. Mircheff for their valuable time and
thoughtful insights and suggestions.

My greatest appreciation and gratitude to my mentor, Dr. Maria Edman, who has
made my journey of the past two years highly valuable and enjoyable, teaching me how
to be independent in my thinking, encouraging me to give my best and for always being
there.

A special thanks to Francie Yarber for always helping me for the smallest things,
and to Gerald Nagatani and Srikanth Janga who helped me with the techniques when I
was just starting out. I am also grateful to all the other members of my lab that have made
my experience fun-filled and worth cherishing. Thank you; Janette Contreras, Lilian
Chiang, Eunbyul Evans, Kaijin Wu, Ben Xu, Daniel Diaz, Aaron Hseuh, Hua Pei, and
Guoyong Sun.

Finally, I would like to thank my family and friends for their constant support and
encouragement throughout my graduate studies.

iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
ABBREVIATIONS vi
ABSTRACT viii
CHAPTER 1: Studies of Lacrimal Gland Immune Cell Homeostasis
in Polymeric Immunoglobulin Receptor (pIgR)
Knockout Mice 1
Chapter 1: INTRODUCTION 1
Chapter 1: BACKGROUND 3
1.2.1 Dimeric IgA and Transport by pIgR 3
1.2.2 Ocular Immunity 11
1.2.2.1 Components of EALT 12
1.2.2.2 Forms of EALT 16
1.2.2.3 Types of Immune Response 20
Chapter 1: METHODS 23
1.3.1 Animals and Experimental Groups 23
1.3.2 Schirmer’s Thread Test 24
1.3.3 Fluorescein Staining of the Cornea 24
1.3.4 Lacrimal Gland Collection 25
1.3.5 Light Microscopy and Histological studies 25
Chapter 1: RESULTS 26
1.4.1 Inflammation is seen in pIgR KO mice lacrimal glands 26
1.4.2 TMZ treatment does not reduce inflammation in pIgR
KO mice 28
Table 1: Average number of foci containing more than
10 cells 28
1.4.3 Basal tear secretion is not altered in pIgR KO mice 31
1.4.4 pIgR KO mice do not show higher levels of corneal
damage 33
Chapter 1: DISCUSSION 35
Chapter 1: CONCLUSION 39

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CHAPTER 2: Evaluation of Monoclonal Dimeric IgA producing
Rabbit Hybridomas 42
Chapter 2: INTRODUCTION 42
Chapter 2: MATERIALS AND METHODS 45
2.2.1 Generation of Hybridomas 45
2.2.2 Protein Concentration 46
2.2.3 Western Blotting 47
Chapter 2: RESULTS AND DISCUSSION 49
2.3.1 Molecular Characterization of Rabbit dIgA and pIgA
and presence of J-chain 49
2.3.2 Screening of Hybridoma supernatants for dimeric IgA 52
Chapter 2: CONCLUSION 55
REFERENCES 57

v

LIST OF FIGURES
Figure 1: Transcytosis by pIgR of polymeric immunoglobulins 6
Figure 2: Functions of sIgA and pIgR 10
Figure 3: Eye Associated Lymphoid Tissue (EALT) 19
Figure 4: Lacrimal gland inflammation in WT and pIgR KO mice 27
Figure 5: Lacrimal gland inflammation in WT for TMZ treated
and untreated mice 29

Figure 6: Lacrimal gland inflammation in pIgR KO for TMZ treated
and untreated mice 30

Figure 7: Schirmer’s Test Results 32
Figure 8: Fluorescien Staining of the cornea 33
Figure 9: Fluorescein Staining Results 34
Figure 10: Molecular characterization of rabbit dIgA and pIgA 51
Figure 11: Primary Screening of hybridoma clones 53
Figure 12: Secondary screening of supernatants of subclones 54

vi

ABBREVIATIONS
APC: Antigen-presenting cells
BSA: Bovine Albumin Serum
CALT: Conjunctiva-associated lymphoid tissue
EALT: Eye-associated lymphoid tissue
EDTA: Ethylenediaminetetraacetic acid
ELISA: Enzyme-linked immunosorbent assay
dIgA: Dimeric IgA
DMSO: Dimethyl sulfoxide
DTT: Dithiothreitol
FBS: Fetal bovine serum
GTP: Guanosine Tri-phosphate
HEV: High-endothelial venules
HRP: Horseradish peroxidase
IEL: Intra-epithelial lymphocyte
Ig: Immunoglobulin
IgA: Immunoglobulin A
IgG: Immunoglobulin G
IgM: Immunoglobulin M
IL: Interleukin
KO: Knock-out

vii

LDALT: Lacrimal drainage-associated lymphoid tissue
LG: Lacrimal Gland
LGAC: Lacrimal gland acinar cells
LPL: Lamina propria lymphocyte
MALT: Mucosa-associated lymphoid tissue
MHC: Major-histocompatibility complex
NOD: Non-obese diabetic
PAGE: Polyacrylamide gel electrophoresis
PBS: Phosphate Buffered Saline
pIgA: Polymeric IgA
pIgR: Polymeric Immunoglobulin Receptor
RT: Room temperature
SC: Secretory component
SDS: Sodium Dodecyl Sulfate
sIgA: Secretory IgA
TLR: Toll-like receptors
TTBS: Tween-Tris Base Saline
WT: Wild-type

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ABSTRACT
Polymeric immunoglobulin receptor, known as pIgR is a characteristic entity of
the transcytotic process, being the receptor most widely studied. Its significance is further
enhanced by its important biological function, as it transports antibodies, especially
dimeric IgA across the epithelium into secretions playing an essential role in the body’s
mucosal immune system. Our interest lies in the eye and the lacrimal gland. Since it is
already established knowledge that the tears and ocular mucosa contain dimeric IgA as
the predominant antibody, we attempted to study an animal model system lacking in this
transporter, the pIgR knockout mouse model, in order to ascertain its importance and
influence in the function of the lacrimal gland and tear fluid. The level of infiltrating
immune cells was increased in the lacrimal gland when pIgR was knocked out, although
the exact nature of the inflammation still needs to be determined. These studies showed
that absence of pIgR may have an effect on the ocular immune system homeostasis.
In addition to studies on pIgR, our attention was also focused on obtaining a
source of rabbit dimeric IgA of a general subclass which acts as a tool to further study the
transcytotic machinery involving pIgR and the various proteins interacting with it during
transcytosis in lacrimal gland acinar cells. For this purpose, B-cell hybridoma cell lines
were generated commercially and the clones were screened using western blotting to
identify the ones appearing positive for dimeric IgA. This method demonstrated for the
first time, the ability to clone ocular antibody-producing cells.

1

CHAPTER 1

STUDIES OF LACRIMAL GLAND IMMUNE CELL
HOMEOSTASIS IN POLYMERIC IMMUNOGLOBULIN
RECEPTOR (pIgR) KNOCKOUT MICE

Chapter 1: INTRODUCTION

The ocular surface in all aspects of its architecture is unique. The anatomy of the
eye is specialized to allow vision and is one of the most exposed non-keratinized surface
of the body, yet its immune response is dormant. The ocular surface is said to possess
characteristics of an immune privileged tissue as its immune system is inclined towards
non-responsiveness (30, 112). One of the most prevalent protective factors of the eye
surface is immunoglobulin A (IgA). The varied functions of IgA make it competent to
counter external pathogens and protect the ocular surface by promoting anti-
inflammatory responses.

IgA in its dimeric form is predominant in all mucosal tissues, including the ocular
mucosa. Here it exerts its effect as secretory IgA (sIgA) which is a complex formed
between dimeric IgA (dIgA) and the secretory component (SC), the cleaved extracellular

2

portion of the transcytotic receptor, polymeric immunoglobulin receptor (pIgR). The
pIgR plays a fundamental role in the transport of immunoglobulins (Igs) such as dIgA,
polymeric IgA (pIgA) or pentameric IgM across the epithelial membrane and into
secretions. Thus, this receptor is essential for immune protection at the mucosal surfaces.
Absence of pIgR alters normal immune response and intracellular trafficking
mechanisms. Our studies are based on this receptor and its contribution to the lacrimal
gland (LG), its secretory product, the aqueous layer of the tear fluid, and ocular immune
protection.

To study this further, we analyzed the difference between LG histology in pIgR
knockout (pIgR KO) mice and C57BL/6 (Wild-type WT) mice. In preliminary findings it
was seen that there was increased infiltration of immune cells in the pIgR KO mice. This
effect could have been due to internal immunological changes triggered by the receptor
being knocked out, or due to a higher susceptibility to infection. In order to determine
whether the changes in immune infiltration were due to the influence of conventional
external pathogens, the two strains of mice were bred under special care with the
administration of a broad spectrum antibiotic combination, sulphamethoxazole and
trimethoprim (TMZ). This investigation attempted to ascertain whether the observed
inflammation was due to an autoimmune mechanism associated with the absence of an
active transporter or instead due to a residual infection caused by microorganisms such as
bacteria. However, our data showed that treatment with TMZ did not seem to produce a

3

reduction in the inflammation relative to the untreated controls. Further studies are
required to quantify and better understand the causes behind these changes.

Chapter 1: BACKGROUND

1.2.1. Dimeric IgA and Transport by pIgR
The adaptive immune system is broadly divided into cellular and humoral defenses,
with the former protecting against intracellular invasions and the latter against
extracellular pathogens. The humoral arm involves antibodies which are present in the
blood and mucosa, each possessing different characteristics (83). Since we are concerned
with the ocular surface which is an established mucosal membrane, our focus was the
mucosal antibodies, of which IgA is the most prevalent Ig and is credited with the first
line of specific defense at these surfaces (60, 83, 127). It is produced and secreted locally
by plasma cells residing in the connective tissue underlying the epithelium. Plasma cells
are activated with the help of T-cells along with cytokines or by exposure to the antigenic
proteins expressed on cell surfaces of antigen-presenting cells (APCs) via Major
histocompatibility complex (MHC) class II molecules (29, 58, 59). T-cells are activated
when their receptors interact with the MHC-presented antigenic fragments. They in turn
directly contact B-cells via binding of specific cell surface receptors which results in T-
dependent B-cell activation. However, certain bacterial proteins such as

4

lipoplysaccharides can directly activate B-cells in a T-cell independent mechanism (6, 7,
37, 96).

IgA exists in both serum and mucosal secretion albeit in different polymerized forms.
90% of the IgA in serum is monomeric whereas the external secretions are saturated with
pIgA, primarily dIgA (14, 19, 60, 127). dIgA consists of two monomeric subunits bridged
by a peptide called J-chain with disulphide linkages. Just before its release from the
plasma cells, the J-chain is incorporated into pIgA and IgM (pentameric), becoming a
distinguishing feature of all polymeric forms of Igs (25, 49, 90). The J-chain plays an
important role in regulating polymerization and is indispensable for binding of dIgA with
its receptor, pIgR, as it creates an interactive site in the polymeric Igs permitting direct
binding (16, 88). However, it is not absolutely necessary. Studies with J–chain deficient
mice have shown reduced levels of IgA in external secretions and high concentrations of
circulating monomeric IgA. Although significant, these outcomes were seen with hepatic
transport of IgA, in contrast to the intestinal and nasal secretions which appeared to be
independent of J-chain (45, 46, 52). In general, J-chain-containing polymers show high
affinity for binding pIgR and are transported across the epithelium into secretions by
transcytosis.

pIgR is an integral membrane glycoprotein with a ligand-binding extracellular
domain, a single transmembrane region and a cytoplasmic domain (91). It is synthesized

5

by mucosal epithelial cells in the rough endoplasmic reticulum and sent to the trans Golgi
network where it is sorted and delivered by vesicles to the basolateral surface. Here it
binds only the oligomeric forms of IgA and IgM present locally in the mucosa. pIgR,
whether loaded with its ligand or unloaded, is endocytosed and delivered to early
basolateral endosomes where it is packed into vesicular compartments and then sorted
into apical endosomes to be transcytosed to the apical membrane (91, 98). Upon reaching
the apical surface, the extracellular ligand-binding portion of pIgR, called SC is
proteolytically cleaved. This severed peptide fragment is either released as free SC or
remains covalently bound to dIgA by a single disulphide bond to the J-chain forming
sIgA (20, 91, 95, 97). Therefore, pIgR is responsible for transport of IgA from the lamina
propria across the mucosal epithelium into luminal secretion, such as saliva, milk, tears,
bile and other gastrointestinal, reproductive, and respiratory secretions (13, 15). (Fig 1).

6

Fig. 1: Transcytosis by pIgR of polymeric immunoglobulins
pIgR is produced in the endoplasmic reticulum (ER) and delivered to the trans-Golgi
network (TGN) where it is sorted to the basolateral membrane. pIgR then binds to dIgA
and IgM secreted by the locally present plasma cells. This complex is endocytosed and
packaged into the basolateral early endosomes (BEE) to be sorted to the apical endosome
(AE) via a common endosome (CE). At the apical surface, the extracellular part of pIgR is
cleaved off while remaining attached to dIgA, which is secreted as complex called
secretory IgA (sIgA) into the lumen. Unbound pIgR travels through the same pathway and
forms unbound or free secretory component (SC) at the apical surface.
(Adapted from 55, 91)

7

Studies with pIgR KO mice have shown greatly reduced concentrations of sIgA in
intestinal and hepatic secretions and a 100-fold increase in serum IgA as compared to
normal mice (51, 110, 119). However, a significant amount of IgA was still found in
exocrine secretions (110). Interestingly, the serum IgA was found to be predominantly
polymeric, indicating that probably due to the lack of active pumping of dIgA into
secretions, it instead accumulated in the mucosa, and was poured into circulation via
paracellular routes (51). The reason for an increase in dIgA could also be attributed to the
3-fold increase in IgA-secreting plasma cells in the intestinal lamina propria of pIgR KO
mice as compared to the wild type mice. These studies suggested that pIgR might
contribute to mucosal B-cell homeostasis along with mediating transport of dIgA.
However, no difference was seen in the CD8
+
cells in the lower respiratory tract of the
pIgR KO mice as compared to wild-type mice after induction with influenza virus,
indicating that anti-viral cytotoxic T-cell responses are not affected with the absence of
sIgA in the respiratory tract (119). Similarly IgA KO mice were protected against
influenza virus infection if they were previously immunized by viral vaccines, suggesting
that sIgA is not necessary for antigen-induced immunity in mucosal tissues (84).

Function of secretory IgA and pIgR
The normal mucosal architecture is constantly challenged by various environmental
factors, including invading pathogens such as bacteria, viruses, parasites. Non-specific
defense is executed by the innate immune system, where as the adaptive immune system

8

delivers a targeted counter-attack comprising of B and T-lymphocytes and Igs (55, 77,
103).

sIgA is the most effective amongst the mucosal Igs such as IgM and IgG which
mount the first line of specific defense (41, 77, 128). It has enhanced stability due to the
bound SC which prevents its degradation (79). IgA delivers a three-tiered defense at the
lumen, lamina propria and intracellularly. After being transcytosed by pIgR, sIgA forms a
protective barrier at the mucosal lumen that mediates immune exclusion of antigens and
foreign matter as well as prevents the attachment of these external pathogens at the
epithelial surfaces (55, 83, 93, 97, 126). In addition, it can also neutralize the toxicity of
the bacterial products (120). Alternatively, IgA in the dimeric form is involved in
neutralization of intracellular microorganisms such as viruses, within the epithelium. If it
encounters an internalized virus as it is trafficking through the transcytotic pathway, it
can inhibit its assembly or release from the cell (55, 81, 82). In the lamina propria, the
pathogens that have breached the epithelial barrier are encountered by dIgA that is
continuously secreted by the native B-cells. This excretory function employs pIgR as a
major effector due to its ability to trap the antigen-dIgA immune complexes which are
formed and subsequently transporting them across the epithelium into the external
secretions to be eliminated by the body. This restricts colonization and infection by
microorganisms of the mucosa and entry of the pathogen into systemic circulation thus
preventing diseases (56, 83). (Fig. 2).

9

pIgR KO mice have reduced sIgA leading to compromised epithelial barrier function,
as evidenced by increased albumin levels, reflecting leakage of serum proteins at the
mucosal surface-lining fluids such as saliva and feces. Serum IgG levels were also
increased which could be the result of the elevated serum IgA levels competing with its
catabolic degradation. It could also be attributed to the systemic immune response
skewing towards IgG in the absence of sIgA which was seen with its consequent increase
due to development of antibodies to E. coli (51). In addition, pIgR KO mice were also
shown to be susceptible to mycobacterial infection in the respiratory tract (115)
suggesting undue induction of the systemic immunity and destruction of immunological
tolerance of the mucosal barrier against normal antigens. Thus pIgR and sIgA are
important in maintaining mucosal homeostasis and its integrity (51, 119).

10

Fig. 2: Functions of sIgA and pIgR
(1) At the lumen: pIgR binds to dIgA and transports it from the lamina propria to the lumen
where it is released as sIgA. sIgA attacks invading antigens (Ag) and mediates immune
exclusion and prevents their adhesion to the epithelial membrane.
(2) Intracellular: dIgA while being transported encounters a virus that was internalized and
prevents its assembly or release from the cell, preventing further infection.
(3) At the lamina propria: dIgA locally present in the mucosa binds to the antigen (Ag)
forming a dIgA-Ag immune complex. This complex binds to pIgR and is transcytosed
across the cell and released into the lumen to be excreted by the body.
(Adapted from 103)

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1.2.2. Ocular immunology (Fig. 3)
The ocular surface consists of the cornea and its supporting tissue, the
conjunctiva. In some cases, this definition may include the ocular mucosal adnexa which
consist of the LG and its drainage system (67). The ocular surface is established as a
mucosal membrane and uses a unique complementary association between innate and
adaptive immune responses typical of a mucosal tissue and additional protection provided
by the tear film and anatomical barriers (41). This immune system of the ocular surface
forms an eye-associated lymphoid tissue (EALT) (66, 67, 68) which functions in a
contrary fashion such that it attacks the invading pathogens as well as acts to limit the
inflammatory response that could be catastrophic for the delicate structure of the eye
(87). The ocular surface is considered to have “immune privilege” (30, 69) due to the
mechanisms employed by the EALT such as immunological ignorance, immune
tolerance and an immunosuppressive local microenvironment. These mechanisms are all
inclined towards non-reactivity and anti-inflammatory responses (67). The LG is the key
producer of dIgA in the tear film which contributes to secretory immunity by forming a
protective layer at the mucosal surface (17, 39, 88). Thus, interaction between cellular
and humoral immunity serves to balance the tolerance between normal ocular flora,
external irritants, the blood and lymph supply to the cornea and inflammatory response.
These complex interrelated processes comprising of the epithelial barrier, tear film,
antigen presenting cells, antigen recognizing receptors and other effector lymphocytes

12

and antibodies prove to be effective in the prevention of external infection. Imbalance
between any of these functions leads to infections and immune-mediated diseases (41).

1.2.2.1. COMPONENTS OF OCULAR MUCOSA: EALT
• Cornea
It is the outermost layer of the ocular surface and is a specialized transparent
tissue. It consists of tightly packed connective tissue sandwiched in between two
layers of epithelia (22). Epithelial cells form a seal due to the existence of tight
junctions which makes the cornea a physical barrier to the entry of external
pathogens. They also produce mucin which forms a layer on the surface and
subsequently prevent attachment of invading microorganisms (5). Under normal
physiological conditions, the normal cornea’s lack of blood or lymph vessels, is
associated with reduced inflammatory responses, making the cornea an immune-
privileged site even though it is constantly exposed to external stimuli (31, 69).
This phenomenon is due to certain active and passive mechanisms such as the
lack of vasculature (angiogenic privilege (39, 57, 112, 123), reduced MHC class
II-positive APCs, specifically dendritic cells and Langerhans cells, and reduced
corneal expression of MHC I (30, 33). Also contributing are the processes of
immune ignorance, immune tolerance and development of an immunosuppressive
environment (67). Toll-like receptors (TLR) are responsible for antigen
recognition and initiation of immune responses. Their absence from the epithelial

13

surface impairs antigen presentation to the APCs thus granting ignorance (67,
117). Tolerance is due to the presence of regulatory T-cells which promote
unresponsiveness (67). Key factors in the development of an immunosuppressive
environment are the presence of soluble factors, such as transforming growth
factor beta and vasoactive intestinal peptide, which cause apoptosis of effector T-
cells, and which also induce antigen-stimulated B-cells to retain expression of the
J-chain but undergo class switch recombination to IgA and avoid class switch
recombination to the inflammatory form isotypes, IgG and IgE. This is of the
utmost significance, because sIgA has anti-inflammatory actions and does not
activate complement factors (67).

• Conjunctiva and CALT
The conjunctiva is the support system of the cornea and consists of a
connective tissue rich in blood and lymph vessels known as lamina propria which
is present under the epithelial layer, separated by a basement membrane. The
Conjunctiva-associated lymphoid tissue (CALT) is a specialized aggregation
consisting of B and T-lymphocytes, plasma cells and dendritic cells which are
involved in specific defense, along with macrophages and neutrophils which are
involved in innate defense (67, 68, 74). The lymphoid cells occur in organized
follicles as well as dispersed throughout the epithelium and lamina propria
underlying the conjunctiva (74). Previously, they were erroneously considered to

14

be inflammatory cells even though plasma cells were detected in every normal
conjunctiva examined (1). Recent research, however, has shown the important
role that CALT plays in preventing inflammatory processes (1, 11, 12). Similarly,
it was discovered that the among the population of plasma cells, IgA-positive
cells are more prevalent than IgG- or IgM-positive cells, indicating that the
conjunctiva is an active site of local sIgA production (74), a function formerly
thought to occur only in the LG. sIgA is secreted into the ocular surface fluid
forming a protective layer (17, 18, 62, 70, 71). In most animal species, there was a
confirmed presence of IgA-producing plasma cells and follicles associated with
CALT. In humans, specialized high endothelial venules (HEV) present in the
conjunctiva regulate lymphoid cell migration and exchange between the ocular
tissue and with other organs, thus incorporating it as part of the body’s mucosa-
associated lymphoid tissue (MALT) system (31, 44, 63, 64, 67, 74). Thus, the
conjunctiva along with the LG plays an important role in maintaining ocular
surface homeostasis by regulating lymphocytes and soluble immune modulators
(74, 75, 113, 125).

• Lacrimal Gland and its Drainage system (LDALT)
The ocular surface, comprising of the cornea and conjunctiva, along with the
LG and its drainage system constitutes a functional immunological unit (66, 67).
Anatomically, the LG is continuous with the conjunctiva via the lacrimal

15

excretory ducts. It is a tubulo-acinar gland, organized into lobes, with short
branched tubules ending in secretory acini (22). The LG possesses its share of
immune cells such as B and T-lymphocytes, dendritic cells, macrophages and
plasma cells, located in the loose connective tissue surrounding the acinar cells.
The majority of B lineage cells are IgA-secreting plasma cells (40). This feature
suggested that the LG is a unit of the secretory immune system. This
interpretation was validated by findings that IgA and its transporter, SC, are
present in normal human LG (2, 40, 125, 131). Similar observations were made in
the LGs of rats and mice (68). Apart from production of antibodies, the LG also
contributes to the innate mechanism as the acinar cells synthesize, store and
secrete a wide array of proteins into the tears, of which many have anti-microbial
properties (132).

The conjunctival mucosa continues into the lacrimal drainage system through
the lacrimal sac and into the nose via the nasolacrimal duct (67). Additionally,
diffuse lymphoid tissue along with MHC class II-positive dendritic cells and
lymphoid follicles are present which makes the lacrimal drainage system a part of
the mucosal system of the eye and is referred to as the lacrimal drainage
associated lymphoid tissue (LDALT) by some investigators (65, 66, 72, 73, 99).
The LDALT can be considered as an immune inductive site and when exposed to
tears carrying antigens can lead to the proliferation of effector cells (73, 74).

16

In summary, it can be said that the conjunctiva and cornea along with the lacrimal
gland and the lacrimal drainage system together constitute the entire immune system of
the eye. Studies have shown that the diffuse lymphoid tissue with the IgA producing
plasma cells and SC is continuous from the periacinar tissue of the LG via the lacrimal
excretory ducts, to the conjunctival mucosa and into the lacrimal drainage towards the
nose (66, 70). Also, since these tissues are functionally connected via the flow of tears
their immune functions can be combined, thus making them all a functional mucosal unit
for immune protection (68).

1.2.2.2. FORMS OF EALT
As described above, the confirmed presence of lymphocytes and plasma cells in
the conjunctiva, lacrimal gland and lacrimal drainage system, has led to the concept of
EALT which is connected to the other mucosal tissues (MALT) via regulated migration
of lymphoid cells (63, 64, 67, 89). In a broad classification, the lymphoid tissue exists in
two forms: organized lymphoid tissue and a diffuse lymphoid tissue.

• Organized lymphoid tissue
Certain lymphoid tissues of the EALT occur as organized follicles which
show typical characteristics in mucosal tissue. They are present in the conjunctiva
and lacrimal drainage system of humans and some animal species but not in rats
and mice under normal conditions (27, 67, 68, 86, 109). The LG on the other hand

17

rarely shows the presence of follicles (40, 69, 125). The follicles, considered as
the afferent arm of the ocular immune system, are an assembly of B-cells with
parafollicular T-cells as well as a rich supply of blood and lymph vessels (57, 75).
Antigens move past the epithelial barrier overlaying the follicles, where they are
phagocytosed by APCs such as Langerhans cells and macrophages and presented
to naïve lymphocytes in the follicles. Lymphocytes whose antigen receptors
recognize antigen epitopes and determinants become activated and are induced to
proliferate (42). These eventually differentiate into effector T-lymphocytes and
dIgA-positive plasmablasts.

• Diffuse lymphoid tissue
The diffuse lymphoid tissue, as its name suggests, comprises a thin,
continuous and inconspicuous layer of lymphoid cells interspersed along the
epithelium and the lamina propria of the conjunctiva and the LG. These cells
include effector B and T-cells which are generated upon exposure of the antigen
to the follicles. They constitute the intraepithelial lymphocytes (IEL) located in
the basal layer of the epithelium, the lamina propria lymphocytes (LPL) residing
in the connective tissue (1, 41, 107) and plasma cells. In the IEL CD8
+

suppressor-cytotoxic T-cells are more frequent than CD4
+
T helper cells, whereas
in the lamina propria, the distribution is reversed (36, 41, 107). CD8
+
cells serve
as immunosuppressants in the ocular environment. The plasma cells produce

18

dIgA which bind to pIgR in the overlying epithelium and are transcytosed to be
secreted as sIgA (68, 74). This tissue is considered to be the efferent arm of the
immune system and is populated by lymphatic and blood vessels. This allows the
migration of the activated cells to other mucosal tissues in the body which return
to their effector organs, that is, the LG and conjunctiva via HEV’s (41, 67, 68, 69,
76).

Although diffuse tissue is present profusely in humans, it is very rarely
mentioned in animals. A mixture of plasma cells and lymphocytes found
interstitially in the LG can also be characterized as diffuse lymphoid tissue where
the former are more frequent and the CD8
+
T suppressor cells dominate over the
CD4
+
T-cells. In addition, cells with similar characteristics accompanied by MHC
class II positive dendritic cells were seen in the lacrimal drainage system (65, 71).
Thus, the diffuse lymphoid tissue extends from the LG along the conjunctiva and
through the lacrimal drainage system down towards the nose (66, 70) and
functions as a immunological effector site for both cellular and secretory
immunity (68).

The lymphoid follicles form an effective system where the antigens can be locally
detected and counter-attacked by providing the ocular tissue with specific lymphocytes or
plasma cells to generate cellular as well as humoral immune responses (68).

19

Fig. 3: Eye Associated Lymphoid Tissue (EALT)
The ocular mucosa is continuous from the lacrimal gland to the conjunctiva ending into the
lacrimal drainage system, thus forming the three components of the EALT. The forms of
EALT are the organized follicles which make up the CALT and LDALT and the diffused
lymphoid tissue which is spread all over the ocular mucosa. The EALT consists of T cells,
B-cells and plasma B-cells which predominantly are IgA producers. These cells exhibit
innate and adaptive immune responses on invasion. The red arrows indicate the flow of
tears and its proteins from the lacrimal gland over the eye surface and drainage into the
nasolacrimal duct. The green and red arrows indicate the vasculature of the ocular mucosa

20

1.2.2.3. TYPES OF IMMUNE RESPONSE
The ocular mucosa is constantly exposed to the external environment, putting it at
a risk of microbial invasion. Even so, its construction is such that it promotes
immunosuppression rather than inflammation. This system is composed of various
proteins and cellular components that act to protect via innate and adaptive responses.
Innate immunity is the first line of defense and is non-specific as compared to adaptive
response which is more specific.

• Innate Immunity
Innate immunity includes anatomical barriers such as mucins and epithelium
along with the anti-microbial proteins of the tears. It aims at detection and non-
specific destruction of microbial pathogens (41). Mucins produced primarily by
the goblet cells of the conjunctiva and the LG coat the ocular surface preventing
bacterial adhesion and colonization (5). The epithelium itself serves as a barrier
due to the presence of tight junctions forming a relatively impenetrable layer (41).
Tear film is secreted by the acinar and ductal cells of the secretory epithelium of
the LG as a complex solution of ions and proteins and additional antibodies such
as IgA are poured into it from the resident immune cells (123). LG-specific
antimicrobial proteins, produced by the epithelial cells of the conjunctiva,
lacrimal sacs and nasolacrimal surfaces, found in tears are lysozyme, lipocalin
and lactoferrin which either destroy the bacterial components or prevent their

21

growth and metabolism (85). Some of the proteins come under the category of
chemokines and cytokines that recruit and activate the immune cells (34, 57, 61,
105, 106). Tear film is a functionally important component of the innate immune
system and plays a pivotal role in preventing invasion.

Bone marrow derived cells such as macrophages and dendritic Langerhans
cells act as APCs and contribute to the innate immunity (67). Mast cells residing
in the lamina propria secrete cytokines which are responsible for the recruitment
of other leukocytes such as neutrophils thus orchestrating an inflammatory
response (41).

Toll-like receptors (TLR) are another important component of this system as it
functions as a trigger as well as a modulator of the immune response. They are
present on cells which are most likely to encounter a microorganism such as
dendritic cells, neutrophils and macrophages. They recognize molecular patterns
on pathogens and can induce phagocytosis, expression of co-stimulatory
molecules as well as secretion of chemokines such as Interleukin-6 (IL) and IL-8
leading to neutrophil recruitment, thus generating an inflammatory response (21,
24, 130). Certain TLRs occurring in human corneal cells are said to create an
immuno-silent condition by not inducing inflammation in response to

22

lipopolysaccharides as a potential mechanism to prevent unnecessary
inflammatory responses to normal bacterial flora at the ocular surface (117).

• Adaptive Immunity
Adaptive immunity represents the second line of defense and is divided into
cellular and humoral immunity mediating their responses via T-lymphocytes and
antibodies produce by plasma B-cells respectively. As compared to the innate
immunity, adaptive immunity confers specificity and immune regulation. An
afferent antigen uptake by APCs and TLRs is followed by degradation through
which the processed antigen is loaded onto MHC class II molecules and
subsequently presented to lymphoid cells instigating a specific response involving
differentiation and proliferation of effector cells forming an efferent distribution
(41). This process of presentation of antigens demonstrates the interplay between
innate and adaptive response. Clonal expansion of the cells into plasma B-cells
usually secreting IgA and T-lymphocytes confers immunological memory making
the defense system highly selective (41). In addition, antigen recognition alone is
not sufficient to activate differentiation; it also requires the influence of cytokines
which are specific for different immune reactions. For instance, IL-4 supports the
production of plasma cells that secrete anti-inflammatory IgA whereas IL-12
promotes inflammatory cytokines such as interferon-gamma (INF-γ) or tumor
necrosis factor-alpha (TNF-α) (67).

23

Chapter 1: METHODS

1.3.1. Animals and Experimental groups
Male and female C57BL/6 (WT) and pIgR

KO mice were euthanized at 12 weeks.
The pIgR

KO breeder mice were a kind gift from Dr. Richard Strugnell at the University
of Melbourne. C57BL/6 breeder mice were purchased from Harlan Laboratories, Inc.
(Indianapolis, IN). Both colonies of mice were bred in the University of Southern
California Vivarium and housed under special conditions including soft bedding
(Carefresh Ultra, Absorption Corp., Bellingham, WA) so as to minimize corneal damage.
Female and male pups of WT and pIgR KO mice were weaned when they were 3 weeks
old and then either treated with TMZ antibiotic (Sulfamethoxazole and Trimethoprim
Oral Suspension, USP; 200 mg/40 mg per 5 ml; HI-TECH PHARMACAL Co,
Amityville, NY) or left untreated until the time of sacrifice. Thus, the experimental
groups of both strains and gender were: (1) pIgR KO treated with TMZ, (2) pIgR KO not
treated with TMZ, (3) C57BL/6 treated with TMZ and (4) C57BL/6 not treated with
TMZ. The drug was given at a dose of 500 μl, three times a week, which was added to a
water bottle protected from light, and placed in a cage containing about 4 to 5 mice. At
12 weeks of age, the mice were weighed and anaesthetized intra-peritoneally with a
mixture of ketaject (55 mg/kg body weight) and xylaject (14 mg/kg body weight) in PBS
and tests were performed as described below, and animals were sacrificed by cervical
dislocation. All procedures were approved by the institution’s Institutional Animal Care

24

and Use Committee (IACUC) and in accordance with the Association for Research in
Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and
Vision research.

1.3.2. Schirmer’s Thread Test
Phenol Red cotton threads (ZONE-QUICK, Showa Yakuhin Kako Co. Ltd,
Tokyo, Japan) were used to evaluate basal tear secretion volume in the four experimental
groups. The thread is impregnated with phenol red which gives it a yellow color (acidic),
and when it comes in contact with tears it changes to a red color. Mice were
anaesthetized as described above. Forceps were used to place the thread in the lateral
canthus of each eye. After 5 minutes, the threads were removed and the wetted length of
the thread was measured in millimeters using a peak scale Lupe.

1.3.3. Fluorescein Staining of the Cornea
Corneal fluorescein staining was conducted to assess the corneal damage. 2 μl of
fluorescein sodium (0.6 mg in 200 μl; Ful-Glo, Akorn Inc, Buffalo Grove, IL) was added
onto the ocular surface. To distribute the fluorescein evenly on the surface, eyelids were
closed manually three times. Staining was assessed using the Motic camera software and
photographed in the dark using a dissection microscope (Motic, Xiamen, China) attached
with a camera (Motic) while the eye was illuminated with a cobalt blue light lamp (Zeiss,
Germany). The photographs were evaluated by dividing the cornea into four parts and

25

counting green fluorescein stained spots in each quadrant. Scores were assigned by
counting the number of grids showing the green spots, with 4 being the highest and 0
(zero) being the lowest score.

1.3.4. Lacrimal gland collection
After completion of the thread test and fluorescein staining, mice were sacrificed
by cervical dislocation for tissue collection. Lacrimal glands were exposed by making an
incision between the eye and the ear. The glands were located and removed carefully so
as to prevent mechanical damage and to ensure that the gland was whole. Once removed,
they were immediately fixed in 10% neutral buffered formalin, then paraffin embedded,
sectioned and stained with hematoxylin and eosin (H & E staining) using standard
protocols.

1.3.5. Light Microscopy and Histological studies
H & E stained sections of LGs from each experimental group were observed for
morphology by wide-field light microscopy (Nikon Eclipse 80i; Optronics; Picture Frame
imaging software). Preliminary semi-quantitative assessment of the inflammation was
done by counting the nodes (>10 cells) of immune cell infiltration in the entire section.
The average number of the foci per gland was calculated for the entire experimental
group. The Microscopy Sub Core at the USC Research Center for Liver Diseases
provided the microscope used for these studies.

26

Chapter 1: RESULTS

1.4.1. Inflammation is seen in pIgR KO mice lacrimal glands
H&E stained LG sections were analyzed by light microscopy. Three to four mice
from each group were sacrificed and their glands collected. Each gland section was
surveyed for infiltrating lymphocytes; the number of foci containing more than 10 cells
were counted and averaged for each gland. Semi-quantitative analysis showed that the
numbers of foci in pIgR KO mice was higher (1.29 ± 1.49 in males and 0.98 ± 1.08 in
females) as compared to WT mice (0.38 ± 0.52 in males and 0.7 ± 0.82). Along with
defined foci, there were diffused lymphocytes present all throughout the gland which
appeared relatively more abundant in pIgR KO than in WT mice. These observations can
indicate that knocking out pIgR causes some changes in the immune induction and
homeostasis of lymphocytes in the LG. These results can be related to the suggested
study that pIgR is involved in B-cell homeostasis in the mucosa (119).

27

Fig. 4: Lacrimal gland inflammation in WT and pIgR KO mice
The inflammation in pIgR KO mice appears to be much greater than in WT mice. Images
were obtained using the 10X objective and the marked area images were obtained using the
20X objective.

28

1.4.2. TMZ treatment does not reduce inflammation in pIgR KO mice
Our intention of administering TMZ antibiotic was to reduce the burden of
conventional external pathogens and test the hypothesis that underlying low level or
residual infection was responsible for the inflammation noted in pIgR KO LGs. For this
purpose, H&E stained LG sections of 12 week old mice for all four experimental groups
were analyzed. TMZ did not decrease the number of foci in either pIgR KO or WT mice
(see Table 1). Likewise, TMZ treatment did not cause any change in LG morphology.

Table 1: Average number of foci containing more than 10 cells.

Strain Gender
Number
of
glands
Number
of mice
Treatment
Average number of
foci per gland
(>10 lymphocytes)
C57BL/6 Male
4 2
Untreated 0.38 ± 0.52
C57BL/6 Male
6 3
TMZ 1.33 ± 2.06
pIgR KO Male
5 3
Untreated 1.29 ± 1.49
pIgR KO Male
8 4
TMZ 1.44 ± 3.33
C57BL/6 Female
7 5
Untreated 0.7 ± 0.82
C57BL/6 Female
7 4
TMZ 0.0 ± 0.0
pIgR KO Female
6 4
Untreated 0.92 ± 1.08
pIgR KO Female
6 3
TMZ 0.50 ± 0.90

29

Fig. 5: Lacrimal gland inflammation in WT for TMZ treated and untreated mice
Blue arrows show major areas of inflammation and the black arrow heads show diffused
inflammation. Due to technical problems, some the sections were not very distinguishable,
this could probably be due to damage to the gland during preservation.

30

Fig. 6: Lacrimal gland inflammation in pIgR KO for TMZ treated and untreated mice
Blue arrows show major areas of inflammation and the black arrow heads show diffused
inflammation. Due to technical problems, some the sections were not very distinguishable,
this could probably be due to damage to the gland during preservation.

31

1.4.3. Basal Tear secretion is not altered in pIgR KO mice
The thread test was performed on 12 week old male and female mice of all four
groups. Comparisons were made between pIgR KO and WT mice as well as between
TMZ treated and untreated mice for pIgR KO and WT controls (n = 2 to 6 mice per
group). Student’s T-test indicated that there was no statistically significant difference
between the volume of tears secreted by pIgR KO and WT mice. Similarly, there was no
significant difference between TMZ treated and non-treated groups.

32

Fig. 7: Schirmer’s Test Results
Basal tear secretion for each experimental group was measured. Graphs show comparison of
the average tear secretion
A. Shows comparison between the TMZ treated versus the non-treated mice of both genders
of the pIgR KO mice and the C57BL/6 mice (n = 2 to 6 mice).
B. Shows comparison between pIgR KO and C57BL/6 mice of both genders which have
either been treated with TMZ or left untreated (n = 2 to 6 mice).
C57: C57BL/6; pIgR: pIgR KO; UNT: Untreated; TMZ: TMZ treated

33

1.4.4. pIgR KO mice do not show higher levels of corneal damage
Fluorescein staining is a preliminary test done to see the damage on the cornea.
Student’s T-tests revealed no significant differences between fluorescein scores of pIgR
KO and WT mice (n = 2 to 5 mice per group) or between TMZ treated and non-treated
pIgR KO mice (n = 4 to 6 mice per group).

Fig. 8: Fluorescein Staining of the cornea
Pictures were taken of the eye after adding fluorescein. For scoring, the eye was
divided into four grids as shown and the green stained spots were assessed (n = 4 to
6 mice per group).

34

Fig. 9: Fluorescein Staining Results
Experimental mice were instilled with fluorescein dye in each eye and assessed for corneal
damage. Graphs show comparison of the fluorescein score assessed for each eye separately.
A. Shows comparison between pIgR KO and WT mice of both genders (n = 2 to 5 mice).
B. Shows comparison between the TMZ treated versus the non-treated mice of both genders
of the pIgR KO mice and the WT mice (n = 4 to 6 mice).
WT: C57BL/6; pIgR: pIgR KO; UNT: Untreated; TMZ: TMZ treated

35

Chapter 1: DISCUSSION

Our objective was to analyze the role of pIgR in the LG and the ocular mucosa.
pIgR is an important component of the secretory immune system as it is involved in the
generation of sIgA. Preliminary studies in our laboratory showed that pIgR KO mice had
infiltrating cells around the ductal areas of their LG. Previous studies have shown that
pIgR KO mice are more susceptible to mycobacterial infection in the respiratory tract (4,
115). Thus, to reduce the influence of bacterial infection, we raised the mice post-
weaning on an antibiotic. TMZ was used due to its effectiveness against a wide variety of
microbes, namely bacterial infections. As controls, C57LB/6 mice were used and
subjected to similar treatment and breeding conditions.

pIgR KO mice showed higher numbers of foci of infiltrating cells when compared
with WT mice and this effect was prevalent even after treatment with TMZ. In Sjögren’s
syndrome, increasing lymphocytic infiltration reflects the expansion and activation of
CD4
+
helper T-cells (89, 100). As discussed, the numbers of IgA producing plasma cells
are three-fold greater in the intestinal lamina propria of pIgR KO than WT mice, thus
suggesting that pIgR is involved in B-cell homeostasis (119). FACS analysis, previously
performed in our lab revealed all immune cells are increased in pIgR KO mice,
especially, B and T-cells (data not shown). An alternative hypothesis to the origin of the
increased immune cells is that a decrease in the amount of sIgA at the ocular surface,

36

caused due to pIgR KO, is sensed by a feedback mechanism. In principle, such a
mechanism could detect the sIgA concentrations directly or indirectly, that is; this
decrease would either allow a different spectrum or a larger load of microbes to access
the ocular surface. This would lead to increased antigen presentation to the organized
lymphoid tissue, which in turn could activate the differentiation and proliferation of IgA-
producing plasmacytes.

Treatment with TMZ did not show a significant difference in the numbers of
infiltrating foci between pIgR KO and WT mice or amongst the treated and untreated
groups. These findings lead to the hypothesis that LG inflammation in pIgR KO mice is
not a response to external bacterial pathogens, but is, rather, intrinsic. If it is intrinsic, it
might be induced by the lack of pIgR or may be autoimmune in nature. Autoantigens and
alloantigens could be generated and subsequently presented to T-cells by APCs. T-cells
could then activate B-cells to produce antibody (6, 7, 96, 41, 43). The greater population
of dIgA-producing plasma cells in the LG could also be directed against microbial
antigens without an implication towards autoimmunity. Another possible explanation for
the failure of TMZ to influence lymphocytic infiltration of the LGs is that since TMZ is
an anti-bacterial, it did not protect against viral infections. This would suggest that the
inflammation observed in pIgR KO mice could be due to viral infection.

37

Although, the difference is not significant, the female mice showed a reduction in
the number of infiltrating foci, whereas surprisingly, the male mice seem to be showing
an increase for the same. Studies have shown that male rats have more dIgA-producing
cells than females because of the presence of testosterone (9, 10, 114, 116). It is possible
that the gender specific hormone differences could amplify the response induced by the
lack of pIgR seen in the male mice.

The findings that cotton thread tests scores did not differ between pIgR KO and
WT mice suggest that acinar cell physiology related to transport of ions and water is not
affected by the absence of pIgR. However, this does not exclude the possibility that the
content and the quality of the tear fluid might have been affected. Previous studies have
shown that lymphocytic infiltration destroys LG acinar cells, thus majorly affecting tear
output as seen in some dry eye syndrome patients and animal models (32, 89). That our
observations revealed no decrease in tear production might indicate that the lacrimal
infiltration observed in the pIgR KO mice is of a different character than seen in dry eye
models such as the Non-obese diabetic (NOD) mouse (108). Further research is required
to determine the nature of inflammation and infiltrating cells in the pIgR KO mice.

pIgR KO and WT mice showed no difference in corneal fluorescein staining and
TMZ treatment did not produce any change either. We used special soft bedding to
prevent additional mechanical damage to the cornea so as to make the measurement more

38

reliable and attributable to a specific cause if applicable. Further tests and studies need to
be performed to assess histological changes in the cornea which may occur due the
absence of sIgA from the ocular surface fluid. Interestingly, our investigation so far has
detected higher levels of IgA and IgG in tears (data not shown) and if the antibody is
dimeric, that could probably account for the normal cornea. This data could also indicate
the possibility of a mechanism by which the lacrimal epithelial cells can transport dIgA
and IgG into the tear film without pIgR.

Further analysis is also required to verify the presence of the infiltrating cells in
then LG of pIgR KO mice. Also, it is appropriate to determine if there is an increase in
the number of cells over a period of time by assessing the LGs of pIgR KO mice at
different ages. A possible method can be to count the total number of infiltrating
lymphocytes independently, without considering the foci alone. Alternatively the size of
foci or area occupied by the foci relative to the entire area of the gland can be determined.

39

Chapter 1: CONCLUSION

pIgR and sIgA have an established role in the immune protection of the ocular
surface mucosa. Studies have been carried out with pIgR KO mice to probe further into
the mechanism of pIgR transcytosis and more importantly its contribution to secretory
immune system.

The numbers of infiltrating foci were higher in pIgR KO mice that the WT mice
and this was seen even after treatment with TMZ. This finding leads us to suggest the
hypothesis that LG inflammation in pIgR KO mice is not in response to a natural
infection, but rather it is intrinsic. However, the possibility of lymphocytic infiltration
due to viral infections cannot be completely ruled out as was shown in previous studies in
which pIgR KO mice exhibited reduced protection against infection with influenza A and
B (8, 129). Thus, it would be interesting to use immunostaining for IgA and IgG
producing plasma cells, B-cells, CD8
+
cytotoxic-suppressor T-cells, CD4
+
helper T-cells
and APC’s in the lacrimal gland and conjunctiva to elucidate the type and class of
immune cell infiltrates as well as the cytokines associated with them.

Thread tests revealed no significant difference between pIgR KO and WT mice.
This finding suggests that acinar cell function was not compromised. However, some
modest changes have been revealed by proteomic studies of the tear film, which showed

40

increased albumin levels as well as increased IgG and IgA antibody levels (data not
shown). In addition, treatment with TMZ did not alter the volume of tear secretion.

Fluorescein staining tests were conducted to assess possible corneal damage. The
cornea is an immune-privileged site which is predominantly protected from external
pathogens by sIgA. Damage to the cornea can allow penetration of the pathogen resulting
in abrogation of immune tolerance and initiation of an immune response. There was no
change in the fluorescein scores of the WT and pIgR KO mice. Administration of TMZ
did not improve and/or protect the cornea.

These two tests were performed to obtain preliminary data. Previous studies have
shown elevated levels of dIgA in serum and reduced concentrations in mucosal secretion.
The appearance of dIgA in the mucosal secretions of pIgR KO mice was probably due to
paracellular transport. The passive traffic of antibodies to the lumen has been proposed as
the reason that the intestinal lumen showed no histological evidence of inflammation
(51). However, the LGs of pIgR KO mice showed increased lymphocytic infiltration.
Interestingly, our investigation also detected higher levels of IgA and IgG in tears (data
not shown). There are several possible explanations for this finding, including
deterioration of the epithelial barrier due to the absence of sIgA, increased lymphocytic
infiltration, or epithelial irritation. Clarification of whether the IgA detected in the tear
fluid is monomeric or polymeric will give an estimate of the epithelial tight junctions

41

being faulty or the presence of an alternative mechanism by which IgA is transported into
exocrine secretions.

Further studies are definitely required to assess the changes seen in the LG in
pIgR KO mice and to assess the associated immune responses. Determining which
immune cells are present and which cytokines are altered may give insights into the
nature of inflammation as well as the role pIgR and sIgA play in the immune protection
of the ocular surface.

42

CHAPTER 2

EVALUATION OF MONOCLONAL DIMERIC IgA
PRODUCING RABBIT HYBRIDOMAS

Chapter 2: INTRODUCTION

Hybridomas, a revolutionary discovery by Köhler and Milstein in 1975, have
been used for decades for the production of monoclonal antibodies generated against
particular targets. A hybridoma is an immortal cell line developed by fusing a specific
antibody-producing B-cell with a myeloma (B cancer) cell capable of replicating almost
indefinitely and secreting an unlimited quantity of only a single antibody. Our interest
was in obtaining a constant source of rabbit dIgA for research purposes. This dIgA need
not be specifically targeted against any particular antigen. Our approach was to obtain a
rabbit dIgA-secreting hybridoma cell line.

dIgA is the most abundant immunoglobulin in mucosal and exocrine secretions,
whereas in the serum it makes up only 10% of the total IgA, the rest of it being
monomeric IgA (4, 127). Immunoglobulins are produced and secreted by B-cells and

43

plasma cells; they are composed of two heavy and two light chains linked together by
disulphide bonds. The light chains interact with the amino portion of the heavy chains to
give rise to the variable Fab region which contains the antigen binding site. The terminal
domains of the heavy chains together constitute the Fc portion, which interacts with
receptors expressed on immune cells, other cell types, and complement factors to
orchestrate subsequent responses (103). Most of the IgA in the serum is produced by the
plasma cells residing in the bone marrow and spleen (4). In contrast, dIgA is produced
and secreted locally, by the plasma B-cells lodged within the mucosal tissues. These not
only synthesize the monomeric unit (160 kD) but concomitantly produce a cysteine rich
polypeptide (16 kD) called J-chain which links the subunits by disulfide bonds to
generate dimeric and polymeric forms (pIgA) (53, 102, 103, 111, 121). Although, J-chain
is not necessary for the synthesis of polymeric forms, its presence is indispensable for
binding to pIgR, which is the principal receptor implicated in the active transport of dIgA
across mucosal epithelia and into the exocrine secretions (20, 50).

pIgR is expressed by epithelial cells which line the mucosal membrane of the
lacrimal, salivary, and mammary glands, as well as gastrointestinal, respiratory, hepatic
and urogenital tracts (88, 118). It is a transmembrane receptor with a cytoplasmic domain
and an extracellular domain. The extracellular domain binds ligands such as dIgA, pIgA
or pentameric IgM at the basolateral membrane. It internalizes its bound ligands and
proceeds to transport them to the apical surface where the extracellular portion, called

44

SC, is cleaved off with the ligand still attached. When the ligand is dIgA, the product is
sIgA (as discussed in Chapter 1, section 1.2.1). sIgA, released into the lumen, plays a role
in the immune defense at the mucosal surface, as it prevents attachment of invading
organism to the mucosa and also causes their neutralization (26, 38, 49, 55, 83). sIgA is
present in mucosal secretions such as tears, saliva and breast milk. It has been shown to
be produced within the LG and to be present in relatively high proportions in the tears of
rabbit, rats and humans. Thus the LG constitutes a part of the secretory immune system
(3, 38, 39, 101).

Transcytosis is an established process which is involved in the trafficking of
proteins across epithelial cells. The roles of dIgA and pIgR, in this process have been
intensively studied. Mucosal surfaces are constantly being exposed to the external
environment and the first barrier to attacking pathogens is the epithelium. Any damage to
it initiates an immune response mediated by B and T-cells. Along with the cellular
response, a number of antibodies, including IgA, IgM and IgG are also produced and
secreted from both systemic and local lymphoid tissues (28, 35, 55, 102). Transcytosis is
the process that transfers the antibodies across the epithelium and into the lumen, where
they approach the attacking pathogens (55, 91, 92, 103). (Fig. 2 from Chapter1).

We are interested in the trafficking and uptake processes occurring in the LG and
intend to direct our research towards further elucidating the transcytotic machinery. Thus,

45

the interstitial cells from rabbit lacrimal glands were isolated and sent to a company that
owns a rabbit myeloma and offers hybridoma production services. The intention of this
screening was to identify and select clones which were specific producers of dIgA. The
hybridomas were delivered as frozen cells which are currently being thawed and
expanded in our laboratory.

Chapter 2: MATERIALS AND METHODS

2.2.1. Generation of Hybridomas
3 female New Zealand White rabbits (approximately 4 kg body weight) obtained
from Irish Farms (Norco, CA) were first anesthetized with 5 mg of xylamine and 50 mg
of ketamine given in the thigh muscle. They were then euthanized with Euthanasol given
in the ear vein in accordance with the Guiding Principles for the Use of Animals in
Research and a protocol approved by the institution’s Institutional Animal Care and Use
committee (IACUC). The LGs were collected and minced with scalpel blades, digested
with a mixture of collagenase, DNAse and hyaluronidase and rinsed with Hank’s/EDTA.
To isolate the interstitial cells, all washes were collected and spun at 1800 rpm for 5
minutes. Pellets were re-suspended with 10% DMSO in FBS (>10 million lymphocyte
cells). The re-suspended lacrimal interstitial cells were slowly cooled to -80
o
C in a
cryobox and sent to Epitomics Inc. (Burlingame, CA). Epitomics conducted hybridoma

46

fusions between the interstitial cells and myeloma cells in 10 x 96 well plates and
performed custom ELISA screening of these plates using goat Anti-Rabbit IgA as the
coating antibody and goat Anti-Rabbit IgG conjugated with HRP as the detection
antibody at 1:10000 dilution, both purchased from Abcam (Cambridge, MA). All
hybridoma clone supernatants showing appropriate specificity for dIgA were expanded
and confirmed positive by standard ELISA. The hybridoma culture supernatants (30
positive multi-clone supernatants, 1ml for each) were sent to our laboratory for additional
screening. These were screened specifically for dIgA by western blotting using non-
reducing gels and distinguishing dIgA from IgA by molecular weight. From the clones
appearing positive for dIgA, three showing the strongest bands were selected. Epitomics
further expanded the positive clones to produce ten to twelve subclones per clone. These
were confirmed by standard ELISA and their supernatants (1 ml for each) were sent for
further screening. Similar procedures were followed and nine positive subclones were
selected. These subclones were then expanded and delivered to our laboratory as frozen
cells for further use.

2.2.2. Protein Concentration
Protein concentrations were determined for each supernatant. Each was diluted
1:50 using PBS. For the first 30 clones, a 96 well plate was loaded in duplicates with 10
μl of the above dilution, 15 μl of deionized water and 150 μl of Biorad dye (Bio-Rad
Laboratories, Inc., Hercules, CA) diluted 1:5 with deionized water. Standard solutions of

47

0, 0.5, 1.0, 1.5, 2.0 and 2.5 μg Bovine Serum Albumin (from a 200 μg/ml BSA solution)
were prepared in PBS. 25 μl of the standards were loaded in duplicates and 150 μl of the
diluted dye was added to each well. For the supernatants of the subclones, similar
procedure was carried out using 7.5 μl of the 1:50 diluted samples, 17.5 μl of deionized
water and 150 μl of Biorad dye. The plate was incubated at room temperature for at least
5 minutes after which the absorbance was read using a GENios Plus multi label reader
(TECAN, Crailsheim, Germany; Magellan software, TECAN Austria, GmbH) at 570 nm
and compared with the standard BSA curve. Average protein concentration was
calculated for each supernatant.

2.2.3. Western Blotting
Supernatants of the clones and subclones were screened using SDS-PAGE
followed by immunoblotting to analyze the polymerization state of IgA. A 6% non-
reducing, denaturing (0.1% SDS) polyacrylamide gel was prepared for separation with a
4% stacking gel. Samples equivalent to 75 μg of protein were diluted with PBS and
mixed with 1 volume of 4X SDS-PAGE loading buffer (8% SDS, 40% glycerol, 0.26mM
Tris-HCL, pH 7.8, 0.4% bromophenol) without dithiothreitol (DTT). Samples were
boiled for about one minute to denature them. 20 μl were loaded into each well and the
gel was run for 4 hours and 30 minutes at 4
o
C. 5 μg of purified Human IgA1
lambdadimer (1 μg/μl, Nordic Immunological Laboratories, The Netherlands; United
Sates distributor, Accurate Chemicals and Scientific Corp, Westbury, NY) and 75 μg of

48

rabbit tears were used as positive controls. Molecular weight markers (HiMark
TM
Pre-
Stained High Molecular Weight Protein Standard, Invitrogen, CA) were run to enable
estimates of protein size. The first 40 minutes of the gel were run at 85 V and the
remaining time at 130 V. The fractioned proteins were blotted onto a nitrocellulose
membrane (BioTrace™ NT, Pall Corporation, Pensacola, FL) at 40 V overnight for 12
hours at 4
o
C. After transfer, the membrane was blocked using blocking buffer (Rockland,
Gilbertsville, PA) for an hour with shaking at room temperature (RT), and then incubated
with primary antibodies overnight at 4
o
C or for minimum 3 hours at RT. The primary
antibodies mixture contained: goat polyclonal Anti-Rabbit IgA (1 mg/ml; Abcam,
Cambridge, MA), 1:1000 and mouse monoclonal Anti-Human J-chain IgG1 (200 μg,
ThermoScientific-Pierce, Rockford, IL), 1:500 diluted with blocking buffer. The
membrane was washed 4 times for 15 minutes each with TTBS and then exposed to the
appropriate dye-conjugated secondary antibodies mixture, which contained donkey Anti-
Goat IgG (IRdye800; 1 mg/ml; Rockland, PA); 1:2000 and goat Anti-Mouse IgG
(IRDye700DX; 1 mg/ml; Rockland, PA); 1:2000. Secondary antibody incubation was for
60 minutes at RT. The membrane was washed again with TTBS and scanned at 700 nm
and 800 nm with a Li-Cor Odyssey Infrared Fluorescence Imaging System (Lincoln,
NB).

49

Chapter 2: RESULTS AND DISCUSSION

The purpose of the B-cell hybridomas was to produce rabbit dIgA which could
then be used for various uptake and transport mechanistic studies. Thus the criterion for
selection of clones was that they should be definite producers of dIgA rather than only
monomeric IgA. The resulting clones were sent to Epitomics who expanded the positive
subclones and sent the final product as frozen hybridomas, which could now be
developed and grown in the laboratory to supply rabbit dIgA.

2.3.1. Molecular characterization of rabbit dIgA and pIgA and presence of
J-chain
Initially, to characterize the polymerization state of IgA and presence of J-chain,
denaturing 6% polyacrylamide gels were used under non-reducing conditions to run
rabbit tears, serum and human tears along with human dimeric IgA1 as a positive control.
In the dimeric and polymeric forms, individual IgA molecules are linked by disulphide
bonds and by an additional cysteine rich J-chain polypeptide (111). Non-reducing
conditions were used to prevent the cleavage of these disulfide bonds (50). These
conditions were created by preparing a loading buffer without adding DTT. Heating was
carried out to cause denaturation. Analysis of fractions observed after immunoblotting
showed the different species separated as monomeric, dimeric and polymeric based on
their molecular size. Polymeric IgA was observed as the uppermost band followed by

50

dIgA at about 400 kD. Monomeric IgA migrated further down to about 170 kD. Usually
two bands were observed at dIgA position, with the likelihood that the higher molecular
weight variants were polymeric forms of IgA (Fig. 10). This was distinguished clearly in
the human dimeric IgA1 control. Similarly, in some cases two bands were seen near the
monomeric form, the higher one being a J-chain positive monomeric IgA migrating
slightly slower than the ordinary monomers (50). These bands were more prominent in
the hybridoma samples (Fig 10).

A small amount of sample did not enter the gel under non reducing conditions
probably due to the extremely large size of the antibody or it could be due to the
formation of insoluble aggregates or large covalent complexes that formed during
preheating (53).

The membranes were scanned at two different wavelengths, 700 nm for detecting
J-chain and 800 nm for IgA. Scanning at 700 nm showed that only the polymeric, dimeric
and J-chain positive monomeric IgA along with dIgA control contained J-chain, whereas
the purely monomeric form was not detected. Remarkably, due to the conservation of the
J-chain among various species; amino-acid homology (identity) between human and
rabbits being 86% (73%), there was cross reactivity. Thus the cross-reactive mouse
antibody against human J-chain and goat antibody against rabbit IgA could be used
effectively for immunoblotting with the hybridoma samples and control human dIgA.

51

Fig. 10: Molecular characterization of rabbit dIgA and pIgA
A. Human dIgA was separated on a non-reducing polyacrylamide gel and blotted with
an anti-J-chain and anti-IgA antibody to observe the different polymerization states.
The uppermost band is possibly pIgA, followed by dIgA at approximately 400 kD. J-
chain is detected at 700 nm and can be seen in dIgA and pIgA. Monomeric IgA is seen
at approximately 170 kD. The higher band is assumed to be J-chain associated IgA
which is clearly seen.
B. Human dIgA was blotted along with rabbit tears (Rt) and rabbit serum (Rs) to
determine the size of dIgA. Bands are seen at approximately 400 kD indicating the
presence of dIgA. Rabbit tears do not show distinct bands as the proteins seem to have
degraded on storage. Rabbit dIgA was observed to run slightly lower than the human
dIgA, probably due to the variation in different species.
L: Molecular weight marker
B
700nm
800nm
L dIgA dIgA Rt Rs
460

268
238

171

75
A
700nm 800nm

pIgA

dIgA

mIgA

L dIgA dIgA
460

268
238

171

75

52

2.3.2. Screening of hybridoma supernatants for dimeric IgA
Protein concentrations of the supernatants of the clones and subclones were
determined using the protein concentration assay. Western blotting was employed for
screening of all the samples under non-reducing conditions using a 6% denaturing SDS-
PAGE to identify the clones producing dIgA. In the primary screening of the clones, after
probing with antibodies, each sample was compared with the control human dimeric
IgA1 and rabbit tears and also amongst themselves. Selection of a clone identified
positive for dIgA was made on the basis of molecular size, intensity of the band and
detection of J-chain. From 30 clones, 7 were chosen which showed bands similar to the
control and had relatively intense bands as compared to the other supernatants. It was
observed that rabbit dIgA ran slightly lower than the human protein, at approximately
350 kD. This may be due to variation among species which leads to a difference in the
molecular size. Also, the rabbit tears used as control, did not give distinct bands, which
could possibly be due to degradation of the proteins during storage. The selected samples
were once more analyzed by western blotting to allow for a more precise comparison
amongst them. Three positives from these, samples numbers 13, 15 and 31, were selected
as the most appropriate clones (Fig. 11). These three clones were then expanded by
Epitomics and the supernatants of the subclones were sent for a secondary screening.
Similar procedures and selection parameters were employed and finally nine subclones
were selected from a total of 34 samples (Fig 12).

53

Fig. 11: Primary Screening of hybridoma clones
6% polyacrylamide gels were used under non-reducing conditions to screen the
supernatants of the clones as described. Human dIgA (dIgA) and rabbit tears (Rt) were
used as controls. Membrane was probed with a mixture of antibodies against rabbit IgA
and human J-chain. The reactive bands were observed for the presence of dIgA. A
primary screening was carried out with all the supernatants. The blot shows 7 most
positive samples which were chosen and analyzed again. Samples # 13, 15 and 31 were
eventually selected based on the intensity of the band, presence of J-chain and
appropriate molecular size as compared to dIgA control. Rabbit tears do not show
distinct bands as the proteins seem to have degraded on storage. Rabbit dIgA from the
hyridomas was observed to run slightly lower than the control human dIgA, at
approximately 350 kD, probably due to the variation in different species Bands seen at
approximately 170 kD are monomeric IgA which may be produced due to cleavage of
the disulphide bonds or due to denaturing caused by heating or presence of SDS. The
three positive clones are marked by asterix (*), clone #15 being strongly positive for
dIgA.
L: Molecular weight marker

L dIgA Rt 1 3 10 13 15 31 37

460

268
238

171

75
pIgA

dIgA

mIgA
* * *

54

Fig. 12: Secondary screening of supernatants of subclones
Similar western blotting procedures were used to screen the supernatants of the
subclones of sample# 13, 15 and 31. Here only subclones of 15 and 31 are shown. At
800 nm, all samples show distinct bands for presence of dIgA at the appropriate
molecular size, with subclones of sample #15, being the strongest. J-chain can be seen
quite clearly in all the samples when scanned at 700 nm. Molecular ladder is not visible
at 800 nm.
L: Molecular weight marker
700nm

460

268
238

171

dIgA
L dIgA 15.1 15.4 15.11 15.12 31.2 31.3 31.8
800nm
L dIgA 15.1 15.4 15.11 15.12 31.2 31.3 31.8
31.12
dIgA

460

268
238

171

55

Chapter 2: CONCLUSION

The machinery involved in the process of transcytosis has been a very popular
area of research since many years. It is now considered a principal mechanism for the
delivery of proteins from the basolateral to the apical surface, thus adeptly maintaining
the polarity of epithelial cells (94). The transcytosis of dIgA via the polymeric
immunoglobulin receptor has been notably studied for this phenomenon. In the
transcytotic secretion of dIgA, an extracellular portion of the receptor is cleaved off,
forming the SC that remains bound to dIgA to form sIgA, which plays an important role
in the defense of the mucosal surfaces exposed to pathogens (28).

Production of dIgA-secreting hybridoma cells allows us to isolate and purify dIgA
and use it for specific studies involving its trafficking and uptake machinery in vitro.
New proteins are being discovered that are involved or recruited by pIgR during
transcytosis. For example, pIgR transcytosis is regulated by cytoskeleton proteins (47,
80) such as Rho GTPases (54, 78, 104), Rab GTPases such as Rab3b, Rab3D, Rab11,
Rab17 and Rab25 (23, 38, 48, 122, 124) and SNARE proteins along with stimulation of
transcytosis by ligand binding (103). Research in this arena provides a large scope for the
discovery of novel moieties that may play a role in some step of transcytosis or other
trafficking and signaling pathways. Our studies are focused on the lacrimal gland, thus

56

the LGAC’s can serves as a relevant model system to study the proteins that regulate
transcytosis (38).

A clinically significant area of research includes the use of pIgR as a means of
targeted gene delivery to epithelial cells and the use of sIgA for immunotherapy and other
therapeutic purposes (33, 103). dIgA can be used to conduct research is these areas as
well.

The method of analysis using western blots in a non-reducing environment can be
used to study the polymerization state of the antibodies, especially IgA in the tears, serum
and saliva of other species, for example, in pIgR knockout mice. We intend to use this
method to determine the forms of IgA that are present in the serum as well as exocrine
secretions, which may help us determine and quantify the changes occurring due to
knocking out pIgR.

57

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Abstract (if available)

Abstract Polymeric immunoglobulin receptor, known as pIgR is a characteristic entity of the transcytotic process, being the receptor most widely studied. Its significance is further enhanced by its important biological function, as it transports antibodies, especially dimeric IgA across the epithelium into secretions playing an essential role in the body’s mucosal immune system. Our interest lies in the eye and the lacrimal gland. Since it is already established knowledge that the tears and ocular mucosa contain dimeric IgA as the predominant antibody, we attempted to study an animal model system lacking in this transporter, the pIgR knockout mouse model, in order to ascertain its importance and influence in the function of the lacrimal gland and tear fluid. The level of infiltrating immune cells was increased in the lacrimal gland when pIgR was knocked out, although the exact nature of the inflammation still needs to be determined. These studies showed that absence of pIgR may have an effect on the ocular immune system homeostasis.

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Creator Kothawala, Mubashera (author)

Core Title Studies of lacrimal gland immune cell homeostasis in polymeric immunoglobulin receptor (pIgR) knockout mice and evaluation of monoclonal dimeric IgA producing rabbit hybridomas

School School of Pharmacy

Degree Master of Science

Degree Program Pharmacy / Pharmaceutical Sciences

Publication Date 04/20/2010

Defense Date 03/24/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag dimeric IgA,lacrimal gland,OAI-PMH Harvest,pIgR,rabbit hybridomas

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah F. (committee chair), Mircheff, Austin K. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email kothawal@usc.edu,mubasherak@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2937

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