mousumi rahman qazi - diva portal
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Doctoral thesis from the Department of Biochemistry and Biophysics,
Stockholm University, Stockholm, Sweden
Immunohepatotoxicity of the persistent
environmental pollutants perfluorooctanoate
(PFOA) and perfluorooctane sulfonate
(PFOS)
Mousumi Rahman Qazi
Stockholm 2011
Mousumi Rahman Qazi, Stockholm 2011
ISBN 978-91-7447-381-0, pp. 1-70
Printed in Sweden by US-AB, Stockholm 2011
Distributor: Department of Biochemistry and Biophysics, Stockholm University
Cover illustration: SpringerImages.com
TO MY PARENTS
TO MY FAMILY
List of Publications This thesis is based on the following publications, which will be referred to in the text by their Roman numerals: I. Qazi MR, Abedi MR, Nelson BD, DePierre JW, Abedi-Valugerdi M. Dietary
exposure to perfluorooctanoate or perfluorooctane sulfonate induces hypertrophy
in centrilobular hepatocytes and alters the hepatic immune status in mice. Int.
Immphar. 2010: 10 (11):1420-7.
II. Qazi MR, Nelson BD, DePierre JW, Abedi-Valugerdi M. 28-Day dietary
exposure of mice to a low total dose (7mg/kg) of perfluorooctanesulfonate
(PFOS) alters neither the cellular compositions of the thymus and spleen nor
humoral immune responses: Does the route of administration play a pivotal role in
PFOS-induced immunotoxicity? Toxicology. 2010; 267(1-3):132-9.
III. Qazi MR, Bogdanska J, Butenhoff JL, Nelson BD, DePierre JW, Abedi-
Valugerdi M. High-dose, short-term exposure of mice to perfluorooctanesulfonate
(PFOS) or perfluorooctanoate (PFOA) affects the number of circulating
neutrophils differently, but enhances the inflammatory responses of macrophages
to lipopolysaccharide (LPS) in a similar fashion. Toxicology. 2009;
262(3):207-14.
IV. Qazi MR, Xia Z, Bogdanska J, Chang SC, Ehresman DJ, Butenhoff JL, Nelson
BD, DePierre JW, Abedi-Valugerdi M. The atrophy and changes in the cellular
compositions of the thymus and spleen observed in mice subjected to short-term
exposure to perfluorooctanesulfonate are high-dose phenomena mediated in part
by peroxisome proliferator-activated receptor-alpha (PPAR). Toxicology. 2009;
260(1-3):68-76.
V. Blom KG*, Qazi MR*, Matos JB, Nelson BD, DePierre JW, Abedi-Valugerdi M.
Isolation of murine intrahepatic immune cells employing a modified procedure for
mechanical disruption and functional characterization of the B, T and natural
killer T cells obtained. Clin Exp Immunol. 2009; 155 (2):320-9.
*These authors have contributed equally to this paper.
These articles are reprinted here with permission from the publishers.
Additional publication and manuscript:
Qazi MR, Abedi MR, Nelson BD, DePierre JW, Abedi-Valugerdi M. Characterization of
the Hepatic and Splenic Immune Status and Immunoglobulin Synthesis in Aged Male
Mice Lacking the Peroxisome Proliferator-Activated Receptor-Alpha (PPAR). Scand J
Immunol. 2011;73(3):198-207
Qazi MR, Nelson BD, DePierre JW, Abedi-Valugerdi M. Effects of short-term dietary
administration of perfluorooctanoate or perfluorooctane sulfonate and subsequent
withdrawl on hematopoietic cells in the bone marrow of mice. Manuscript
Abstract
Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), manufactured
for a variety of industrial and consumer applications, are ubiquitous environmental
pollutants. Their accumulation in humans and wildlife raises serious health concerns.
Here, we examined the potential effects of PFOA and PFOS on the innate
immune system in mice. Short-term dietary exposure to high doses reduces the total
number and subpopulations of circulating white blood cells. Moreover, production of
proinflammatory cytokines by macrophages in the peritoneal cavity and bone marrow,
but not in the spleen following exposure to in vitro or in vivo stimulation by bacterial
lipopolysaccharides is enhanced. With respect to adaptive immunity, PFOS reduces the
total numbers of thymocytes and splenocytes and subpopulations thereof in a dose
dependent fashion. Furthermore, comparison of wild-type mice and the corresponding
knock-out strain lacking peroxisome proliferator-activated receptor-alpha revealed that
these immunological changes are partially dependent on this receptor. Our further studies
also show that sub-chronic dietary exposure to an environmentally relevant dose of PFOS
does not alter the cellularity of the thymus and spleen and exerts no influence on humoral
immune responses.
To facilitate examination of the effects of PFOA and PFOS on the hepatic
immune system, we developed a procedure for mechanical disruption that yields a larger
number of functionally competent immune cells from this organ. In our last study, lower
doses of PFOA or PFOS induced hypertrophy of hepatocytes and altered the hepatic
immune status. Thus, we find that short-term, high- and low-dose exposure of mice to
these fluorochemicals is immunohepatotoxic.
Sammanfattning
Perfluorooktanat (PFOA) och perfluorooktansulfonat (PFOS) som tillverkas för
många olika industri och konsumentprodukter, är globalt förekommande miljögifter.
Deras ackumulering i människor och djur ger upphov till en stark oro för hälsoproblem.
Vi har granskat effekterna av PFOA och PFOS på det medfödda, ospecifika
immunförsvaret. Exponering för höga doser via maten under kort tid minskar det totala
antalet cirkulerande vita blodkroppar samt delpopulationerna. Immunsvaret ökar dock
efter stimulering med bakteriella lipopolysaccharider både in vitro och in vivo, dvs
produktionen av proinflammatoriska cytokiner av makrofager i bukhålan och benmärgen,
men inte i mjälten ökar. När det gäller adaptiv, specifik immunitet minskar PFOS det
totala antalet tymocyter och splenocyter och deras olika subpopulationer. Vid exponering
för lägre doser av PFOS induceras hepatomegali utan att påverka tymus eller mjälten. Vi
kunde visa att peroxisomal proliferator-aktiverad receptor-alfa medierar effekterna utav
PFOS i tymus samt delar av effekterna av PFOS i mjälten genom att använda möss som
saknade denna receptor. Detta stöds av vår studie med subkronisk exponering för en
miljömässig dos av PFOS vilken inte ändrade den cellulära sammansättningen i vare sig
tymus eller mjälte och inte hade något inflytande på det humorala immunsvaret.
För att underlätta studier av hur PFOA och PFOS påverkar immunsystemet i levern
utvecklade vi en metod för framrening av immunceller via mekanisk sönderdelning av
levern, vilket gav ett större antal av funktionella immunceller från detta organ. I vår sista
studie kunde vi påvisa att lägre doser av PFOA eller PFOS inducerade hypertrofi av
hepatocyter samt en påverkan av leverns immunförsvar.
Contents 1. Introduction ................................................................................................11
1.1 General background ..............................................................................11 2. The immune system ...................................................................................11
2.1 The lymphoid organs ............................................................................11 The bone marrow .....................................................................................12 The thymus...............................................................................................13 The spleen ................................................................................................14
2.2 The innate immune system ...................................................................14 Neutrophils ...............................................................................................15 Macrophages ............................................................................................15 Natural killer (NK) cells ..........................................................................17 Dendritic cells (DCs) ...............................................................................17
2.3 Adaptive immunity ...............................................................................18 2.3.1 Cell-mediated immunity ....................................................................18
Helper T cells ...........................................................................................18 Cytotoxic T cells ......................................................................................19 T cells ...................................................................................................20 NKT cells .................................................................................................20
2.3.2 Humoral immunity .............................................................................21 B cells.......................................................................................................21 T cell-dependent B cell activation ...........................................................22 T cell-independent B cell activation ........................................................22 Immunoglobulins .....................................................................................22
2.4 Cytokines ..............................................................................................23 TNF- IL-6 ..........................................................................................................24 IFN- IL-4 ..........................................................................................................25
3. Liver immunology .....................................................................................25
Kupffer cells.............................................................................................26 Liver sinusoidal endothelial cells (LSEC) ...............................................26 Resident hepatic dendritic cells ..............................................................27 Lymphocytes ............................................................................................27
3.1 Innate immune responses in the liver ....................................................28 3.2 Adaptive immune responses in the liver ...............................................29
B cell responses ........................................................................................29 T cell responses ........................................................................................29
4. The perfluoroalkyl acids PFOA and PFOS ................................................31
4.1 Physical properties ................................................................................31
4.2 Usages ...................................................................................................32 4.3 Global production .................................................................................32 4.4 Distribution in the environment ............................................................33 4.5 Levels in human ....................................................................................33 4.6 Toxic effects..........................................................................................34
Hepatotoxicity ..........................................................................................34 Developmental toxicity ............................................................................35 Immunotoxicity ........................................................................................35 Effects on hormonal levels .......................................................................36
5. Peroxisomes ...............................................................................................37
5.1 Peroxisomes proliferators .....................................................................37 5.2 Peroxisomes proliferator-activated receptors (PPARs) ........................37 5.3 Gene activation by PPARs ....................................................................38
PPAR PPAR PPAR/
5.4 PPARs and effects on the liver .............................................................40 5.5 The roles of PPARs in immunity and inflammation .............................40
6. Present investigations .................................................................................42
6.1 Specific aims .........................................................................................42 7. Methodological considerations ..................................................................43
7.1 Animals .................................................................................................43 7.2 High performance liquid chromatography-mass spectroscopy-mass spectroscopy (HPLC-MS-MS) ....................................43 7.3 Flow cytometry .....................................................................................43 7.4 In vitro cell culture and stimulation ......................................................43 7.5 Assay of cell proliferation .....................................................................44 7.6 Propidium iodide (PI) and the Annexin-V assay ..................................44
8. Results ........................................................................................................45
Paper I .........................................................................................................45 Paper II ........................................................................................................47 Paper III ......................................................................................................48 Paper IV ......................................................................................................49 Paper V........................................................................................................50
9. Concluding remarks ...................................................................................52 10. General discussion and future perspectives .............................................53 11. Acknowledgements ..................................................................................56 12. References ................................................................................................59
Abbreviations
APC Antigen-presenting cell
BCR B cell-receptor
BM Bone marrow
CLP Common lymphoid progenitor
CMP Common myeloid progenitor
CTL Cytotoxic T cell
DC Dendritic cell
G-CSF Granulocyte colony-stimulating factor
HSC Hematopoietic stem cell
IFN- Interferon-gamma
IHIC Intrahepatic immune cell
IL Interleukin
Ig Immunoglobulin
LPS Lipopolysaccharide
MHC Major histocompatibility complex
MZ Marginal zone
NK cell Natural killer cell
PFAA Perfluoroalkyl acid
PFOA Perfluorooctanoate
PFOS Perfluorooctane sulfonate
PPAR Peroxisome proliferator-activated receptor
ROS Reactive oxygen species
SRBC Sheep red blood cell
SCID Severe combined immunodeficiency
TD T cell-dependent
TI T cell-independent
TGF Transforming growth factor
TNP Trinitrophenol
Th cell Helper T cell
TNF-Tumor necrotic factor-alpha
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1. Introduction
1.1 General background
Immunotoxicology can be defined as the study of adverse effects on the immune
system resulting from occupational or environmental chemicals, inadvertent or
therapeutic exposure to drugs, or, in some instances, biological materials. Studies on both
humans and other animals reveal clearly that the immune system is sensitive to a variety
of factors, and that damage to this system can be associated with morbidity and even
mortality. Regulatory agencies have recognized this fact and testing for immunotoxicity
has become a routine component of safety evaluations for therapeutic agents, biological
agents, and chemicals.
2. The immune system
Historically, the collective and coordinated response of the cells and molecules
that constitute the immune system has been viewed as providing protection from disease
and, more specifically, from infectious disease [1]. We now know that many of the
mechanisms responsible for resistance to infections can respond to non-infectious foreign
antigens and, sometimes, even to endogenous epitopes. There are two fundamentally
different types of responses to invading microbes, i.e., the innate and adaptive immune
responses. Innate responses are activated, within 4 hours after infection or tissue injury,
in attempt to immediately inhibit and control the invading pathogen [2]. The adaptive
immune response takes three to five days to produce sufficient numbers of effector cells
directed towards specific targets and to develop an immunological memory which
recognizes and helps prevent later infection with the same microorganism [3].
2.1 The lymphoid organs
The morphologically and functionally diverse organs and tissues that contribute to
the development of immune responses are divided into primary and secondary lymphoid
organs. The thymus and bone marrow, where maturation of lymphocytes takes place, are
the primary (or central) lymphoid organs. The secondary (or peripheral) lymphoid organs,
where lymphocytes interact with antigens, include lymph nodes, the spleen, and various
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mucosal lymphoid tissues, including bronchus-associated lymphoid tissue (BALT) and
gut-associated lymphoid tissue (GALT) [4].
The bone marrow
The complex bone marrow (BM) acts both as the site of hematopoiesis and a fat depot.
After birth and throughout life, cells of the innate and adaptive immune systems are
generated from the hematopoietic stem cells (HSCs) in the mammalian BM (Figure 1)
[5], originating primarily from two separate progenitors, i.e., common myeloid
progenitors (CMP) and common lymphoid progenitors (CLP) [6]. Under the influence of
the microenvironment provided by stromal cells, CMP can further develop into either
megakaryocyte-erythrocyte progenitors (MEP) or monocyte-granulocyte progenitors
(MGP).
Figure 1. Hematopoietic cell differentiation in the bone marrow (adapted from [5]).
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These MEPs can subsequently progress to become erythrocytes or megakaryocytes, while
further differentiation of MGP eventually produces granulocytes (neutrophils, eosinophils
and basophils) and monocytes.
On the other hand, in the presence of stromal cells and the cytokines they
produce, CLP can give rise to mature B and natural killer (NK) cells. This development
to mature B cells proceeds through several stages (i.e., pro-B, pre-B, immature B and
mature B cells), each of which is characterized by the expression of distinct cell surface
molecules, including the B cell antigen receptor, IgM [6-8]. Although the precursors of
T cells also originate from HSCs in the BM, development of these cells is completed in
the thymus [9].
The thymus
The thymus, the site of T cell development and maturation, is a flat, bilobed organ
situated above the heart. Each lobe is organized into two compartments: the outer cortex
is densely packed with immature T cells, called lymphocytes; while the inner medulla is
sparsely populated with thymocytes [10].
T cell differentiation and selection in the thymus is a multi-step process: the entry
of lymphoid progenitor cells; generation of CD4+CD8+ (cell surface marker express on
immature T cells) thymocytes in the outer cortex; positive selection of these double-
positive thymocytes; negative selection of CD4+CD8+ cells and interaction between
positively selected thymocytes and medullary thymic epithelial cells, to complete the
development of the thymocytes and ensure central tolerance; and, finally export of the
mature either CD4+ or CD8+ T cells from the thymus [11].
This multi-step process entails strict selection, during which only 1-3 % of all the
original thymocytes survive. The initial development of the lymphoid progenitors into
mature T cells occurs via a pathway involving expression of CD25 and CD44 and
proceeds until a developmental checkpoint is reached. At this point only those cells that
have succeeded in rearranging the gene encoding the -chain of the T-cell receptor
(TCR)are selected for further differentiation [12].
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The spleen
The spleen is the largest peripheral lymphoid organ in both primates and rodents,
but, in contrast to other such organs, lacks afferent lymphatics and is supplied by the
splenic arteries. The spleen is divided into the red pulp (which surrounds the sinusoids
and is full of erythrocytes), that filters old or damaged erythrocytes out of the blood, and
the white pulp (containing lymphoid follicles, the marginal zone and the periarteriolar
lymphoid sheath), which is composed of innate and adaptive immune cells. The cells in
the white pulp are organized in a highly specialized manner, optimized for efficient
antigen-specific activation of T and B cells [13]. Follicular B cells circulate between the
blood and secondary lymphoid organs; express IgD at high levels, and participate in
classic adaptive T cell-dependent immune responses. The B cells in the marginal zone are
referred to as innate-like and participate preferentially in T cell-independent immune
responses.
The structure of the spleen enables it not only to remove senescent erythrocytes
from circulation, but also leads to the efficient removal of blood-borne microorganisms
and cellular debris. This function, in combination with its highly organized lymphoid
compartment, makes the spleen the most important site for immune reactivity towards
different types of microbes including fungi [13,14].
2.2 The innate immune system
Most of our encounters with microorganisms do not result in disease. The few
microbes that manage to cross the barrier formed by the skin, mucus, cilia and gastric pH
are usually eliminated by innate immune mechanisms, which commence immediately
upon entry of the pathogen [15]. If phagocytosis cannot rapidly eliminate the pathogen,
inflammation is promoted by the production of cytokines and acute phase proteins. This
early response is not antigen-specific nor does it generate immune memory [15]. Only
when the inflammatory process is unsuccessful in eliminating the pathogen, the adaptive
immune system is activated to produce armed effector cells, a process which requires
several days. The various innate cell populations, such as neutrophils, monocytes,
macrophages, dendritic cells (DCs) and natural killer cells, involved in the phagocytic
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uptake of pathogens and endogenous waste, triggered by, e.g., pattern-recognition,
complement and Fc receptors, are described below [16].
Neutrophils
Neutrophils, together with eosinophils and basophils, belong to the group of
leukocytes referred to as granulocytes, since they contain intracellular granules.
Neutrophils are also called polymorphonuclear (PMN) cells due to the segmentation of
their nucleus into several lobes. These, the most abundant white blood cells, are the first
to accumulate at sites of inflammation.
After approximately five days of proliferation and final maturation in the bone
marrow, mature neutrophils are released into the peripheral circulation [17]. Once
activated in response to acute infection, they migrate to the site of the infection, where
they phagocytize foreign antigens and exert anti-microbial action through the production
of reactive oxygen species (ROS) and release of toxic components of their granules
including -defencin, lactoferrin, cathepsin and myeloperoxidase [18]. Following these
inflammatory responses, neutrophils undergo apoptosis and their debris is processed by
macrophages. Neutrophil trafficking from the bone marrow is regulated by the
granulocyte colony-stimulating factor (G-CSF), which also attenuates neutrophil
apoptosis at local inflammatory sites [19].
Macrophages
Macrophages are a ubiquitously distributed population of mononuclear
phagocytes involved in numerous homeostatic, immunological and inflammatory
processes. Their wide tissue distribution allows them to provide an innate defense against
foreign elements prior to the immigration of lymphocytes. Following stimulation,
macrophages release the soluble factors interleukin (IL)-1 and tumor necrotic factor-
alpha (TNF-, which activate the innate immune system. Macrophages also release IL-6
and all of these proinflammatory cytokines induce an acute phase response, which is
crucial to subsequent activation of adaptive immune responses. Interestingly, activation
of the adaptive immune cells feedback regulates macrophages, as exemplified by the
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production of interferon-gamma (IFN-, originally called macrophage-activating factor,
by activated T cells [20].
Macrophages differentiate from monocytes, which migrate from the peripheral
circulation into tissues, both under physiological conditions and in response to
inflammation [21]. Monocytes develop, as mentioned above, from HSCs in the BM.
During monocyte development, myeloid progenitor cells (referred to as
granulocytes/macrophage colony-forming units) give rise sequentially to monoblasts,
pro-monocytes and, finally, monocytes, which are then released from the bone marrow
into the bloodstream [21]. In response to proinflammatory, metabolic and immune
stimuli, monocytes migrate from the blood, into the bone (osteoclasts), alveoli, central
nervous system (microglial cells), connective tissue (histiocytes), gastroinstestinal tract,
liver (Kupffer cells), spleen and peritoneum in order to replenish long-lived tissue-
specific populations of macrophages [22].
Figure 2. Macrophage heterogeneity (adapted from [23]).
The heterogenous morphology of macrophage populations (Figure 2) [23] is
reflected in their functions and biochemical properties. Their developmental requirements
have been employed to distinguish between classically activated (M1) and alternatively
activated (M2) macrophages. The former are activated through toll-like receptors (TLR)
that bind components of bacterial cell walls and interferon- (IFN-an inflammatory
cytokine); whereas the latter are activated by IL-4 or IL-13. A slightly different
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classification, based on involvement in host defenses, wound healing and immune
regulation, has also been proposed [21].
Natural killer (NK) cells
NK cells are cytotoxic lymphocytes that play an important role in innate
immunity by lysing tumor or pathogen-infected cells. NK cells can also release cytokines,
including IFN-, TNF-, granulocyte-macrophage colony stimulating factors (GM-CSF)
and chemokines. These cells are activated by cytokines, following which they bind to the
Fc portion of antibodies and exert antibody-mediated cellular cytotoxicity that disrupts
infected cells. Regulation of this activity is achieved through so called ``activating and
inhibitory receptors`` that help to differentiate between infected and non-infected cells
[24].
The two major subsets of NK cells in humans are distinguished on the basis of the
level at which they express CD56 (CD56bright and CD56dim, respectively). CD56dim NK
cells are more numerous and excel in cytotoxicity, whereas the CD56bright cells are less
cytotoxic, but more potent producers of cytokines [25,26].
Dendritic cells (DCs)
DCs were first described by Ralph Steinman nearly 30 years ago [27]. They are
professional antigen-presenting cells, specialized in capturing, processing and presenting
antigens to immune cells, such as, T cells [28]. They are distributed throughout the body,
particularly at sites of pathogen encounter, such as skin and mucosal surfaces. Upon
antigen encounter, DCs become activated and thereafter travel to secondary lymphoid
tissues, such as the spleen and lymph nodes, where they stimulate naïve T-cells to
differentiate and proliferate into functionally competent effector T cells.
DCs play an important role in activating and differentiating B cells by interacting
with CD40-activated naïve B cells or by producing cytokines such as IL-12. In the
thymus, these cells also have a crucial function in induction of immunological tolerance
by eliminating T cells reactive towards self-antigens through a process known as negative
selection [29,30]. Both mice and human have two major types of DCs; myeloid DCs
(mDCs) and plasmacytoid DCs (pDCs) [31]. Depending on the type of stimuli and which
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of these subsets they belong to, these cells express various cytokines, such as IFN-, IL-
10 and IL-12, involved in the induction of immune responses.
2.3 The adaptive immune system
Adaptive immunity involves the development of highly defined immune
responses and the formation of memory that enables the body to mount more specific and
stronger immune responses upon reencounter with a pathogen [2]. Two major cell types
are involved in maintaining adaptive immunity: T cells and B cells. These cells recognize
specific structures on pathogens via their different surface receptors and are responsible
for the enormous diversity of the adaptive immune system. B cells recognize antigens
through B cell receptors (BCR) and are primarily responsible for humoral immunity. The
T cells perform cell-mediated immunity through the T-cell receptors (TCR) with unique
specificities dependent on interactions with major histocompetibility complex (MHC)
molecules [32]. The differentiation of the cells of the adaptive immune system is
dependent on cross-talk with cells of the innate immune system, with cytokines acting as
key mediators in this context [33]. T- and B-cells share, as mentioned before, common
progenitor cells developed from HSCs in the BM [34].
2.3.1 Cell-mediated immunity
Effector T cells, generated in response to antigen, are responsible for cell-
mediated immunity. Both activated helper T cells (Th cells) and cytotoxic T cells (CTL)
serve as effector cells in cell-mediated immune reactions. Cytokines secreted by Th cells
can activate various phagocytic cells, enabling them to phagocytose and kill
microorganisms more effectively. Cell-mediated immune responses are important in
defending against intracellular bacteria, viruses, and cancer and are responsible for graft
rejection [1].
Helper T cells
Helper T cells (Th cells) or CD4+ T cells (since they express CD4 on their
surface) play a significant role in directing and regulating adaptive immune responses.
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Activation of Th cells is initiated by and totally dependent on recognition of peptides
bound to MHC class II molecules on the surface of the immune cells known as antigen
presenting cells (APCs), which can be either macrophages, DCs or antigen-specific B
cells. Depending on the effectiveness of immune response and the cytokine environment,
CD4+ T cells can be activated to proliferate and differentiate into Th1, Th2 and Th17
cells.
Th1 cells, which secret IL-2 and IFN- are associated with cell-mediated
immunity and the latter cytokine also stimulates macrophages to eradicate intracellular
pathogens. These same cells also promote inflammation and enhance phagocytosis of
antigens opsonized by antibodies [35]. Th2 cells typically produce IL-4, IL-5, IL-10 and
IL-13, which participate in activation and differentiation of antibody-producing B cells
and play a central role in IgE-mediated allergic disease. These cells also potentiate
elimination of parasitic infection [36]. Th17 cells release IL-17 and IL-22 and are
involved in immune responses that are essential for protection against extracellular
bacteria and fungi as well as in several autoimmune diseases [37].
Upon sensing an antigen, CD4+ T cells produce the key cytokine IFN-, which
stimulates macrophages to produce antimicrobial components, i.e., ROS and reactive
nitrogen intermediates designed to kill the bacteria [33].
Cytotoxic T cells
Cytotoxic T cells or CD8+ T cells are MHC class I restricted. They are primary
immune effector cells involved in combating viral infection, as well as, possibly, in the
elimination of tumor cells and rejection of allografts. Following activation by an MHC
class I:peptide complex on APCs, CTL migrate to the periphery to kill infected cells, a
process crucial to limiting the growth and spread of infectious microbes. The infected
cells display peptide fragments that originate from cytosolic pathogens, in particular
viruses [38]. CD8+ T cells express cytotoxic activity by releasing the contents of their
granules, including perforin and granzyme, and by triggering Fas-mediated
apoptosis [39].
The helper and cytotoxic memory T-cell populations are derived both from naive
T cells after these have encountered antigens and from effector cells following antigenic
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activation and differentiation. They are long-lived, quiescent cells that respond with
heightened reactivity to a subsequent challenge with the same antigen, generating a
secondary response [40].
T cells
Although a minor population in the peripheral blood, T cells that recognize
antigen through a cell receptor on their surface constitute a major population among
intestinal intraperitoneal lymphocytes. Unlike T cells, which recognize processed
antigens associated with MHC molecules, these T cells recognize both natural or
synthetic non-peptide antigens [41,42], such as heat-shock proteins (in mice) or
phosphorylated bacterial metabolites (in humans) respectively, as well as controlling the
integrity of epithelia. γδT cells form an entire lymphocyte system that develops under the
influence of other leukocytes, in the thymus and in the periphery. They demonstrate the
same potent cytotoxic activity as conventional T lymphocytes and, moreover, produce
a variety of cytokines, such as keratinocyte growth factor. Little is known about T cell
activation, but their main role in host immune responses is to coordinate the innate and
adaptive immune responses during the early stage of infection [43].
NKT cells
NKT cells are innate-type lymphocytes that express both NK and T cell markers
such as CD161 (NK 1.1), CD3 and a TCR. These cells are also different from
conventional T cells in that they recognize lipid antigens presented by CD1d (an MHC I-
like molecule expressed by antigen-presenting cells) rather than MHC-restricted peptides.
Antigen-mediated activation of NKT cells induces rapid production of cytokines (within
hours), which is indicative of their potent immunomodulatory function.
The size of the NKT cell population varies considerably among individuals, from
undetectable (< 0.001 %) to 3 % of all the lymphocytes in the peripheral blood [44]. NKT
cells develop in the thymus and, when the TCR that recognizes CD1d is expressed,
diverge from conventional T cells at the double positive (CD4+CD8+) stage [44].
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After migrating from the thymus, NKT cells continue to develop in the periphery
and eventually populate the blood, liver, lymph nodes and spleen. Cells that express
CD1d and are thus able to present antigen to these NKT cells include macrophages, DCs
and B cells [45]. Experimentally, NKT cells are most often activated with alpha-
galactoceramide (-GalCer) or by anti-CD3 antibodies. TCR-mediated activation of NKT
cells results in rapid production of large amount of cytokines (e.g. IL-2, IL-4, IL-6, IL-10,
IFN-, and TNF- etc), induction of cytotoxic activity and up-regulation of costimulatory
molecules [45].
2.3.2 Humoral immunity
The humoral immune response (so-called because the antibodies involved are
found in the ``humours``, or plasma, lymph and tissue fluids) is mediated by antibodies
secreted by the cells of the B lymphocyte lineage or B cells. Upon co-stimulation, e.g., by
another antigen-presenting cell, such as DCs, B cells are transformed into the plasma
cells that secrete antibodies. Th2 cells assist the entire process. Secreted antibodies bind
to antigens on the surfaces of invading microbes, such as viruses or bacteria, and promote
phagocytosis.
B cells
The ectopic germinal centers (i.e., the non-lymphoid organs: the rheumatoid
synovial membrane, thyroid gland, choroid, and lungs) and the lymphoid follicles of
secondary lymphoid organs (such as regional lymph nodes, tonsils, spleen, and mucosal
lymphoid tissues) are classical sites of B cell activation, differentiation and proliferation
[46]. Immature B cells transported via the circulation to the secondary lymphoid organs.
Here, when a specific antigen binds to their BCR, these cells become activated. Usually,
signaling via the B cell co-receptor (CD19, CD21, CD81), T cells participate via CD40-
CD40L interactions and cytokines are also required for this activation [47]. B cells can
function as APCs. B cell activation occurs via two different pathways, T cell-dependent
(TD) or T cell-independent (TI), depending on the properties of the antigen.
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T cell-dependent B cell activation
The TD pathway, which is generally associated with memory formation, includes
interaction between activated CD4+ Th cells and B cells [48]. This interaction takes place
in the outer T cell zone in the secondary lymphoid organs and induces further
proliferation and differentiation of the B cells. These important interactions involve
binding of the antigen, processed and presented in a MHC class II complex by the B cell,
to the TCR. In addition, interactions of co-stimulatory molecules contribute to the
subsequent activation of both B and T cells. These co-stimulatory molecules and their
ligands include CD40:CD40L (CD154), CD28:CD80/CD86 and CD70:CD27 [49,50].
CD40 signaling in B cells is essential for induction of Ig isotype switching and is
associated with the formation of memory.
T cell-independent B cell activation
TI pathways can be activated by polymeric antigens with repeating structures that
induce strong signals due to cross-linking and clustering of BCR molecules [2]. This
pathway can be induced either by TI type-1 (TI-1) or TI type-2 (TI-2) antigens. TI-1
antigens, such as lipopolysaccharide (LPS) or flagellin at high concentrations, induce
polyclonal B cell activation. TI-2 antigens consist of molecules with highly repetitive
structures, such as carbohydrate polymers present on bacterial cell membranes. TI-2
antigens are recognized by specific BCRs, expressed especially by B cell subsets in the
marginal zones (MZ) [51].
Immunoglobulins
Immunoglobulins (Ig) are antigen–binding proteins both present on the B cell
membrane and secreted by plasma cells. When bound to the cell surface, the Ig functions
as a receptor involved in differentiation, activation and apoptosis; while the secreted form
neutralizes foreign antigens and recruits other effector molecules [52]. The Ig molecule
consists of two identical light (L) and two identical heavy (H) chains complexed in a
Y-shape. The assembled molecule contains an antigen-binding site (Fab) along with a
crystallisable fragment (Fc) that interacts with receptors present on neutrophils,
23
monocytes, macrophages and complements. The nature of the heavy chain defines the
isotypes of Ig i.e., IgA, IgD, IgG, IgE and IgM.
IgG is the most abundant isotype, constituting approximately 85 % of the total
serum Ig in humans, probably due to its long half-life [53,54]. This is the predominant
isotype involved in secondary immune responses characterized by high-affinity isotype-
switching antibodies [55]. IgG consists of four subclasses, (IgG1, IgG2, IgG3 and IgG4),
numbered in the order of decreasing serum concentration [56].
Since IgM is the first isotype expressed during B cell development, as well as the
major isotype involved in primary antibody responses, the level of this isotype is
frequently used as a marker of infection [56]. IgM constitutes approximately 10 % of
human plasma Igs and is the second most common isotype in the mucosa (after IgA).
IgM is particularly effective in neutralizing and agglutinating blood pathogens, especially
those residing within cells, thus preventing cell-to-cell transfer [57].
Although the two forms of IgA (IgA1 and 2) account for only about 7-15 % of the
antibodies in human serum, this is the predominant class of antibody in extravascular
secretions [56].
Of all the classes of serum Ig, IgE has the shortest half-life and is present at
lowest concentration, accounting for 0.02 % of the total serum antibodies. This isotype is
associated with hypersensitivity and allergic reactions, as well as with responses to
infection by parasitic worms [56].
2.4 Cytokines
Numerous interactions between the cells of the immune system are mediated by
soluble proteins called cytokines, all of which are produced in small amounts in response
to external stimuli, such as microbes; generally demonstrate a short half-life, and bind to
high-affinity receptors on their target cells. While many cytokines act either on the cells
that produce them (called autocrine action) and/or on adjacent cells (paracine action),
most enter the circulation and act on distant tissues (endocrine hormonal action) [58].
Cytokines are involved in the regulation of both innate and adaptive immunity.
They play an important role in activating macrophages to produce large amounts of other
cytokines, as well as in activating and regulating lymphocytes. Depending on the immune
24
response evoked, cytokines are referred to as Th1- or Th2-associated [59]. The major
cytokines involved in immune regulation are discussed below.
TNF-α
TNF-, synthesized primarily by activated macrophages, is a potent mediator of
inflammatory and immune functions. Circulating levels of TNF- are usually very low
under healthy conditions, but rise rapidly during an infection or inflammation [60]. TNF-
influences the expression of adhesion molecules, as well as chemokines that attract
immune cells to the site of infection. TNF- functions by forming a trimer with either the
TNF receptor 1 (TNFR1) or TNFR2 present on the surface of almost all nucleated cells.
IL-6
IL-6 is a multifunctional cytokine whose inflammatory role depends on whether
there is an ongoing acute immune response or not. This signal is involved in the
development of cells and tissues, as well as a variety of pathological conditions [61]. IL-6
is not produced constitutively, but synthesized by many different types of cell, including
monocytes/macrophages, endothelial cells, fibroblasts, adipocytes and myocytes, in
response to inflammatory stimuli such as lipopolysaccharide (LPS), TNF and IL-1 [62].
IFN-γ
IFN- was first identified in the 1960s, on the basis of its distinctive activity
against Sinbis virus [63]. A number of both innate (e.g., NK cells, NKT cells,
macrophages and myelomonocytic cells) and adaptive (e.g., Th1 cells, CTL and B cells)
immune cells can secrete this cytokine. The Th2-associated cytokine IL-4, together with
IL-10, transforming growth factor- (TGF-, and glucocorticoid down-regulate the
production of IFN- [64].
IFN- is a major product of fully differentiated Th1 cells and plays an important
part in the establishment of an antiviral state, promoting cytotoxicity via both direct and
indirect mechanisms [65]. This mediator can also potentiate peptide-specific activation of
25
CD4+ T cells among APCs via upregulation of the class II antigen-presenting pathway
[66].
IL-4
IL-4, a 15-KDa monomer produced by Th2 cells, basophils, mast cells, and
eosinophils, is involved in allergic reactions and the protective immune response against
helminthes and other extracellular parasites [67,68]. IL-4 is the major stimulus for Th2
cell development; suppresses Th1 cell development; and induces IgE class-switching in
B cells.
3. Liver Immunology
The liver plays a key role in metabolic homeostasis, being responsible for the
metabolism, synthesis, storage and redistribution of nutrients, carbohydrates, fats and
vitamins [69]. It is also the primary detoxifying organ of the body, removing waste and
xenobiotics by metabolic conversion and urinary and biliary excretion. The cell types in
the liver that carry out most of these functions are the parenchymal cells, or hepatocytes,
which constitute approximately 70 % of the cells in this organ. The other 30 % are
hepatic sinusoidal endothelial cells (LSEC), innate immune cells (antigen-presenting
Kupffer cell and dendritic cells), NK cells, NKT cells and adaptive immune cells (T and
B lymphocytes) (Figure 3) [73]. The mean yield of intrahepatic lymphocytes (IHL) from
humans and mice is similar (106 IHL/mg) and indicates that the human liver contains
109-1010 lymphocytes [70,71]. The antigen-presenting cells in the liver are believed to be
crucial for the maintenance of tolerance under non-inflammatory conditions [72,73].
About 30 % of the blood volume passes through the liver every minute and such
tolerance toward endogenous antigens delivered by the blood and the ability to launch
immune responses against pathogens are required simultaneously by this organ. This
balance is made possible by complex interactions between hepatocytes, antigen-
presenting cells and effector cells of the innate and adaptive immune system.
26
Figure 3. Overview of the intrahepatic immune system (adapted from [73]).
Kupffer cells
The Kupffer cells comprise 20% of the non-parenchymal cells in the liver and in
fact, represent the largest pool of macrophages in the human body [74]. These cells are
located within the sinusoidal vascular space in the periportal area, where they clear
endotoxins from the passing blood and phagocytize debris and microorganisms. Kupffer
cells can migrate through the hepatic sinusoid and space of Dissé and in this manner
establish close contact with passing lymphocytes and hepatocytes, respectively [73].
These cells produce a number of regulatory compounds, including nitric oxide, TNF-,
IL-6, TGF- and – and ROS [75].
Liver sinusoidal endothelial cells (LSEC)
Constituting about 50 % of the non-parenchymal cells in the liver, LSECs are
located between the hepatocytes and the blood flow [74]. LSECs express molecules that
participate in antigen presentation, e.g., class I and II MHC and cell surface markers such
as CD40, CD80 and CD86 [76,77]. In a manner similar to DCs (see below), LSECs take
up antigens via receptor-mediated endocytosis and/or phagocytosis [78].
27
Resident hepatic dendritic cells
With their capacity to migrate and perform immunosurveillance, DCs are the most
highly specialized antigen-presenting cells in the liver [79]. In a healthy liver, the
predominantly immature DCs are prepared to capture and process antigens [72]. After
activation upon encountering an antigen, they migrate from the space of Dissé to the
regional lymph nodes, where they present the processed antigen to T lymphocytes [80].
Lymphocytes
The lymphocytes scattered throughout the parenchyma and portal tract of the liver
contain both conventional and unconventional subpopulations (Figure 4) [73]. The
conventional T cells include CD8+ and CD4+ T-cells. More CD8+ than CD4+ cells are
usually present in the liver, whereas the number of the former is higher in the circulation
[73].
The unconventional hepatic T cells include two different populations, i.e., those
that express NK markers (called NKT cells) and those that do not. T cells that express the
T cell receptor (TCR) belong to this later group and represent 15-25 % of all
intrahepatic T cells, thereby constituting the largest population of T cells in the body
[73].
Accounting for as much as 30 % of the intrahepatic lymphocytes, classical NKT
cells are also more abundant in the liver than in other organs [73]. Lipid and glycolipid
components of mycobacterial cell walls, i.e., non-peptide antigen targets, are recognized
by these cells, which are overwhelmingly CD4+ or CD4-/CD8--double negative. The dual
role of NKT cells involves participation in immune responses against tumor cells and
infections and maintenance of tolerance [72,73].
Non-classical NKT cells constitute 30 % of the intrahepatic lymphoid population
and are thereby, once again, more abundant in the liver than in any other organ. Their
migration to and/or expansion in the liver is controlled by the hepatic NK cells. NKT
cells exhibit potent cytotoxic activity against virus-infected and tumor cells and express
receptors that both stimulate and inhibit the activities of NK cells [73].
28
Figure 4. Relative sizes of different populations of intrahepatic immune cells (adapted from [73]).
3.1 Innate immune responses in the liver
As mentioned above, the liver is enriched in macrophages (Kupffer cells), NK
cells and NKT cells, key components of the innate immune system. Activation of Kupffer
cells is triggered by bacterial stimuli, such as LPS and bacterial superantigens. The
cytokines produced by these cells play, in turns, roles in modulating the differentiation
and proliferation of other cells. For instance, in response to physiological concentrations
of LPS, Kupffer cells produce TNF- and IL-10, which down-regulate receptor-mediated
antigen uptake and the expression of class II MHC on LSEC and DCs, thereby reducing
T cell activation [74,81]. Kupffer cells are also known to play important roles in
connection with resistance to primary and secondary infections. The IL-12 and IL-18 they
release regulate NK cell differentiation. Other cytokines, e.g., IL-1, IL-6, TNF- and
leukotrienes, derived from Kupffer cells promote the infiltration and antimicrobial
activity of neutrophils [75].
Upon activation, hepatic NK cells modulate liver injury by balancing local
production of pro-inflammatory (Th1) and anti-inflammatory (Th2) cytokines through
their activating and inhibitory receptors. The inhibitory signals are dominant, i.e., NK cell
activation occurs in the absence of ligands, such as class I MHC, which bind to these
inhibitory receptors [82]. Such activation leads to rapid production of IFN-, which
29
stimulates hepatocytes and LSEC to secrete the chemokine CXCL9 and thereby recruit T
cells to the liver [75].
NKT cells are restricted by CD1, which can be expressed by hepatocytes, as well
as by other antigen-presenting cells such as macrophages, DCs and B cells [83]. The
majority of classical NKT cells are activated by IL-12, which is produced by DCs and
Kupffer cells, and this activation typically results in Fas-dependent cell lysis [84].
NKT cells are also involved in combating both viral and bacterial infections in the
liver [85,86].
3.2 Adaptive immune responses in the liver
B cell responses
The function of hepatic B cells remains relatively obscure, since the number of
these cells in a healthy liver is low and they have been difficult to isolate. When infected
with hepatitis C virus (HCV), the liver behaves as an ectopic lymphoid organ, promoting
the proliferation and differentiation of B cells into antibody-secreting cells within the
germinal centers of intraportal lymphoid follicles [87].
T cell responses
Upon exposure to LSEC-mediated antigen presentation under non-inflammatory
conditions the CD4+ T-cells in the liver participate in hepatic tolerance by secreting IL-4
and IL-10 [88]. An elevated hepatic level of IL-10, which is also produced by CD8+ and
Kupffer cells, alters the chemokine receptors on the dendritic cells, thereby attenuating
migration of these cells to the draining lymph nodes [89].
The immune response to most liver-tropic pathogens involves a strong and
sustained response by CD4+ and CD8+ T cell. In the case of infection with intracellular
pathogens presented predominantly on MHC class I molecules, the effecter functions of
intrahepatic CD8+ T, including the production of Th1 cytokines such as TNF- and INF-
and cytolytic mechanisms, have received special attention. The cytotoxic activity of
CD8+ T cells is mediated by perforin and granzymes [90,91]. CD8+ T cells proliferate
30
only in response to recognition of their antigen, which occurs directly in the liver.
Accumulation of cells undergoing activation-induced cell death (AICD), in particular
CD8+ T-cells, occurs in the liver, indicating that this organ is a site for removal of T cells
[92,93].
31
4. The perfluoroalkyl acids PFOA and PFOS
Numerous xenobiotics disrupt the mammalian immune system and, here, we have
focused on the possible immunotoxic effects of perfluorooctanoate (PFOA) and
perfluorooctane sulfonate (PFOS) (Figure 5) [94]. These compounds belong to the family
of perfluoroalkyl acids (PFAA), with a backbone typically 4-14 carbon atoms in length
and a charged functional moiety (usually carboxylate, sulfonate or phosphonate). The two
most extensively studied PFAAs, PFOA and PFOS, each contain eight carbon atoms [95].
PFAAs are relatively contemporary chemicals, being used only during the past half
century [96] and, despite early indications to the contrary, they were long considered to
be biologically inactive. Today, much research is focused on understanding the
distribution of these compounds in the environment, wildlife, and humans and their
potential deleterious effects on health.
4.1 Physical properties
Covalent carbon-fluorine bonds are among the strongest known. Consequently,
compounds containing perfluorinated hydrocarbon backbones are often stable in air at
high temperatures (in excess of 150o C); nonflammable; resistant to degradation by strong
acids, alkalis, or oxidizing agents; and not subject to photolysis. The stability of these
Figure 5. Molecular structure of PFOA and PFOS (adapted from [94]).
Flurine
Sulphur
Carbon
Hydrogen
Oxygen
Figure 5. Molecular structure of PFOA and PFOS (adapted from [94]).
Flurine
Sulphur
Carbon
Hydrogen
Oxygen
Flurine
Sulphur
Carbon
Hydrogen
Oxygen
32
chemicals renders them practically non-biodegradable and persistent in the environment
[97]. The amphiphilic nature of the PFAAs, with their carbon-fluorine chain imparting
resistance to oil/water and a somewhat hydrophilic acidic head group, probably explains
the extremely low surface tension they provide to solutions.
4.2 Usages
The surfactant properties of PFAAs are ideal for many commercial purposes and
the eight-carbon compounds are among the most effective in this context [98]. As
fluoropolymers, PFOA and PFOS have found more than 200 industrial and consumer
applications, ranging from water-, soil-, and stain-resistant coating for clothing fabrics,
leather, upholstery, and carpets, to oil-resistant coatings for paper products approved for
food contact, electroplating, electronic etching, bath surfactants, photographic
emulsifiers, aviation hydraulic fluids, fire-fighting foams (where the monomers are
actually used), paints, adhesives, waxes, and polishes [99].
4.3 Global production
In the year 2000 the production of PFOS and PFOA were 3500 and 500 metric
tons, respectively. After the major manufacturer of PFOS, 3M Company, phased out
production in 2002, global production of this chemical dropped precipitously to 175
metric tons by 2003 [100]. In contrast, global production of PFOA rose to 1200 metric
tons annually by 2004. The consumption of PFOS in all European Union Countries in
2005 was estimated to be 12.23 tons. Recent data show that China and Japan are
presently the largest producers of PFOS (Figure 6) [101]. In 2006 the U.S. Environmental
Protection Agency initiated a program to eliminate emission and use of PFOA by 2015
[102]. At the same time, other PFAA products are being developed to fill the resulting
commercial void.
33
Figure 6. PFOS production and use in different countries (adapted from [101]).
4.4 Distribution in the environment
In certain Nordic countries, the Netherlands, Spain, Austria, USA and Japan,
PFOA and PFOS have been detected in soils, sediment, waste water and sludge, close to
as well as far from fluorochemical manufacturing plants [103-108]. As recently
documented [109,110] the highest levels of PFAAs are present in the liver of fish-eating
animals, including mink, bottle-nose dolphins, polar bears, and ringed seals, living close
to industrial areas [111,112].
4.5 Levels in humans
PFOA and PFOS have been detected in human blood (whole blood, plasma and
serum), breast milk, liver, seminal plasma and umbilical cord blood [113-118] collected
in countries around the globe, including the United States, Canada, Peru, Colombia,
Brazil, Italy, Poland, Germany, Belgium, Sweden, India, Malaysia, Korea, China, Japan
and Australia. In most cases, occupationally exposed workers and general populations
exhibit serum levels of both PFOA and PFOS of approximately 1-2 ppm and
34
0.01 ppm, respectively. The major routes of human exposure to PFAAs include drinking
water, domestic dust, food packaging and cookware [119-124].
4.6 Toxic effects
The toxicology of PFOA and PFOS has been extensively reviewed [100,125-127].
Repeat-high-dose studies with PFOS in rodents and non-human primates have revealed
reduced body weight, increased liver weight, lowered blood level of cholesterol, and a
steep dose-response curve for mortality [128-130]. A 2-year bioassay, in which
Sprague-Dawley rats were exposed to the high dose of 20 ppm PFOS in their diet,
showed an increase in hepatocellular adenomas [100,131] .
Hepatotoxicity
As noted above, PFOA and PFOS have been associated with hepatomegaly in
rodents (Figure 7) and non-human primates, as well as hepatocellular adenomas,
testicular Leydig cell and pancreatic acinar cell adenomas following two-year exposure of
male, but not female mice to PFOA [132]. Activation of the peroxisomes proliferator-
activated receptor-alpha (PPAR) has been suggested to be involved in tumorigenesis
(primarily in the liver) induced in rodents by a number of non-genotoxic carcinogens. In
rats and mice PFOA and PFOS cause peroxisome proliferation [133-136] and induce
peroxisomal fatty acid -oxidation, enhancing the expression of lipid-metabolizing
enzymes and transport proteins [137-140].
Control PFOA PFOS
Figure 7 . Liver hypertrophy in mice after dietary treatment with PFOA and PFOS.
Control PFOA PFOS
.
35
Developmental toxicity
Exposure to PFOA or PFOS in utero delays the development and reduces
postnatal survival and growth of rodent pups. Moreover, exposure of rats to PFOS during
the last 4 days of gestation is sufficient to reduce neonatal survival [126]. When rodent
dams are exposed to PFOS throughout pregnancy, dose-dependent (1-10 mg/kg)
deleterious effects are seen in the neonates [141]. All rat pups are born alive, pink, and
active with the lowest dose, but with 5 and 10 mg/kg, the neonates become pale, inactive,
and moribund within 30–60 min, and all die soon afterwards.
A similar result was reported by Butenhoff [142], Luebker [143] and their
coworkers for the highest doses of PFOS (10 mg/kg) and PFOA (30 mg/kg) employed in
a two-generation study on rats. Survival improves with lower exposure, being
approximately 50 % with 3 mg/kg. Accordingly, the morbidity and mortality of the
newborn rats appears to be correlated to their body burden of the fluorochemical
(108 ppm PFOS in the serum with the 5 mg/kg dose). Furthermore, morphometric
alterations in the lungs of neonates exposed to PFOS were suggestive of immaturity, but
the failure of rescue agents and presence of normal pulmonary surfactant indicate that the
labored respiration and mortality observed in these pups was not due to lung immaturity
[144,145].
Immunotoxicity
Yang and coworkers [146-148] reported on the immunotoxic potential of high-
dose short-term treatment of mice with PFOA. When given in the diet to male
C57Bl/6 mice for 7-10 days, these fluorochemicals reduce body weight and the size of
the epididymal fat pads, elevate liver weight, induce peroxisome proliferation and cause
atrophy of the thymus and spleen (Figure 8). The numbers of thymocytes and splenocytes
are decreased 90 % and 50 %, respectively, probably due to an inhibition of cell
proliferation in the thymus.
In addition to altering splenic cell numbers, these compounds are
immunosuppressive in both in vivo and in vitro systems upon high-dose exposure
(40 and 30 mg/kg respectively) [146,149]. Even a very low, environmentally relevant
dose (5 mg/kg) of PFOS has been reported to suppress humoral immunity [150]. In
36
addition, Fairely and colleagues [151] demonstrated an elevation in circulating levels of
IgE when PFOA (dermal exposure) and ovalbumin were co-administered to rats.
Recently we have demonstrated that PFOA or PFOS alters hematopoitic cell
development in the bone marrow, where all the immune cells are generated. Both
fluorochemicals reduced the total numbers of different hematopoietic cells belonging to
the lymphoid, myeloid and erythriod lineages in the BM in a dose-dependent manner.
Thus, the immunosuppressive effects of high doses of these fluorinated compounds are
due to impairment of expansion of immune cells at this location. This finding could also
explain the reduced populations of neutrophils and macrophages observed in the
periphery and tissues, respectively.
Effects on hormonal levels
Most recently, several studies have documented depression of serum levels of
thyroxine (T4) and triiodothyronine (T3) in rats (adults, adults during pregnancy and
neonates) exposed to PFOS, although a corresponding elevation in the level of thyroid-
stimulating hormone (TSH) expected to occur through feedback stimulation of the
hypothalamus pituitary (HPT) axis is absent [131,141,143,152]. The mechanism behind
the hypothyroxinemia observed appears to involve enhanced expression of OATP2
(organic anion transporter protein) and MRP2 (multidrug resistance-associated protein) as
a consequence of exposure to PFOS, followed by enhanced uptake and metabolism of
thyroxine. Moreover, several investigations [153-155] have reported that administration
of PFOA to adult male rats for 14 days reduces serum and testicular levels of
testosterone, while enhancing serum levels of estradiol.
Control PFOA PFOS PFOA PFOSControl
PFOA and PFOS induced thymic (left panel) and splenic (right panel) atrophy in mice.
Control
Figure 8.
37
5. Peroxisomes
Peroxisomes are single-membrane cytoplasmic organelles present in all
eukaryotic cells. They are about one-fifth the size of mitochondria and most abundant in
cells of the liver and kidney [156]. These organelles contain more than 60 proteins,
including a wide range of oxidases such as urate oxidate, D-amino acid oxidase,
polyamine oxidase and many more. They are responsible for a variety of metabolic
functions, including -oxidation of fatty-acids, glycogenesis, catabolism of polyamines
and cellular respiration [157-159].
5.1 Peroxisome proliferators
A group of xenobiotics referred to as peroxisome proliferators (PPs), including
drugs (hypolipidemic, analgesics, uricosuric), plasticizers, environmental pollutants and
industrial chemicals cause substantial increases in the numbers of hepatic peroxisomes
together with liver enlargement, particularly in rodents [158]. This phenomenon is
associated with enhanced expression of peroxisomal proteins involved in the oxidation of
fatty acids and other aspects of lipid metabolism [160] and the underlying mechanism
involves so-called peroxisome proliferators-activated receptors. PPs are recognized as
non-genotoxic carcinogens.
5.2 Peroxisomes proliferator-activated receptors (PPARs)
In the 1990´s the first nuclear receptor activated by peroxisome proliferators was
cloned and designated as the peroxisome proliferator-activated receptor (PPAR) [160].
The three isoforms of PPARs known at present are PPAR, PPAR, PPAR. These are
encoded by different genes, expressed differentially in various tissues and have specific
functions [161] (Figure 9). Three related PPAR isotypes have been identified in
vertebrates, including Xenopus, mouse, rat, hamster, and human [162].
38
Figure 9. PPARs isoforms and their functions. 5.3 Gene activation by PPARs
The PPARs belong to the family of steroid/retinoid nuclear receptors that act as
ligand-activated transcription factors [163]. These receptors form heterodimers with the
retinoic X receptors (RXRs) and the resulting PPAR-RXR heterodimer binds to DNA at a
so called DR-1 motif or peroxisome proliferator responsive element (PPREs) with the
consensus repetitive sequence AGGTCA [160]. In the absence of ligand, the dimer
recruits co-repressors, histone deacetylases and chromatin-modifying enzymes, which
results in active repression or silencing of transcription [161]. Ligand binding induces
conformational changes in the PPAR-RXR complex, which leads to release of repressors
and the recruitment of coactivators. These ligand-activated complexes then recruit the
basal transcription machinery, resulting in enhanced gene expression.
PPARs bind components of dietary fat or intracellular lipid metabolites with
relatively low affinity. In their role as lipid sensors, PPARs regulate lipid homeostasis
through the activation of genes whose products are involved in lipid metabolism, storage
and transport. PPARs are also involved in the release of anti-inflammatory
factors [160,163].
39
PPARα
PPAR is expressed at highest levels in metabolically active tissues such as the
liver, heart, kidney, skeletal muscle, brown adipose tissue, and spleen [164]. This
receptor is involved in hepatic, skeletal and cardiac lipid metabolism, cellular fatty acid
uptake, triglyceride (TG) catabolism and glucose homeostasis, as well as exerting anti-
inflammatory effects. Furthermore, PPAR plays a regulatory role in controlling cardiac
metabolic switches and is thus able to prevent cardiac dysfunction. Upon ligand binding,
PPAR can also act as an anti-atherogenic factor by modulating systemic and local
inflammatory responses [164]. The endogenous ligands for PPAR include mono-and
poly-unsaturated fatty acids, eicosanoids, long-chain fatty-acyl CoAs and certain
saturated fatty acids [165,166].
PPARγ
PPAR is expressed at high levels in white and brown adipose tissues, the large
intestine and spleen. This receptor plays important roles in adipogenesis, glucose
homeostasis, and insulin sensitivity [167]. Activation of PPAR leads to up-regulation of
fatty acid-metabolizing proteins, which in turn results in adipocyte differentiation.
Agonists of PPAR have been shown to induce terminal differentiation of normal
preadipocytes and liposarcoma cells in vitro and have been proposed for use as potential
therapeutic agents against liposarcoma [168].
Little is presently known concerning endogenous ligands for PPAR. Metabolites
of prostaglandin D2 and cyclopentanone prostaglandin PGA1 have been suggested to be
potent activators, whereas rosiglatazone and pioglitazone act as synthetic ligands for this
isoform [162,169]. There is also evidence that thiazolidinediones (TZDs), a novel class of
insulin-sensitizing drugs used in anti-diabetic therapy, activate PPAR as well [170].
PPARβ/δ
PPAR is ubiquitously expressed in mammalian tissues and was initially believed
to have a general house-keeping role [171]. However, it has now been shown that PPAR
knock-out mice exhibit altered dermal inflammatory responses, impaired wound healing,
40
defects in myelination and embryonal lethality due to placental defects [172-174].
Prostaglandin A and other eicosanoids are ligands for PPAR [165].
Table 1. Rodent Isoforms of PPARs expressed in different tissues
++++++++++/--γ
-+++++++++β/δ
-+++++++++++α
Adipose tissue
SpleenIntestineKidneyLiverIsoform
Table 1. Rodent Isoforms of PPARs expressed in different tissues
++++++++++/--γ
-+++++++++β/δ
-+++++++++++α
Adipose tissue
SpleenIntestineKidneyLiverIsoform
5.4 PPARs and effects on the liver
PPAR mediates many of the hepatic responses to peroxisome proliferators
including peroxisome proliferation and up-regulation of genes encoding fatty acid
oxidation systems in the mitochondria, peroxisomes and endoplasmic reticulum (ER).
The increased rate of peroxisomal fatty acid -oxidation increases cellular levels of H2O2,
which may result in DNA damage and hepatocyte proliferation and thereby contribute to
the development of liver tumors observed when rodents are treated with PP [175].
Targeted disruption of the PPAR gene in mice has confirmed the involvement of this
nuclear receptor in peroxisome proliferator-induced pleiotropic responses, including
hepatocellular carcinoma [176,177].
5.5 The role of PPARs in immunity and inflammation
PPARs are expressed at high levels in different immune cells. PPAR is
expressed at higher levels in macrophages, where it controls the efflux of cholesterol
[178]. Agonists of PPAR exert anti-inflammatory effects in vascular cells by inhibiting
expression of adhesion molecules and enhancing expression of endothelial nitric oxide
synthetase (eNOS). However, certain in vivo investigations have found proinflammatory
effects of PPAR ligands during endotoximia [179]. PPARs also play a variety of
41
regulatory roles in connection with the innate immune system, e.g., enhancing removal of
apoptotic cells by phagocytosis and regulating the production of antimicrobial peptides
[180].
PPARγ is expressed in monocytes, macrophages, DCs, NK cells and T cells. In
macrophages, this receptor plays an important role in lipid homeostasis. Agonists of
PPARγ also exert anti-inflammatory effects in different model system, where these
compounds inhibit the up-regulation of inflammatory genes by LPS, IL-1β and IFN-γ
[181,182]. This receptor is also involved in promoting apoptosis of lymphocytes through
agonist-induced activation of T cells and, furthermore, activation of PPARγ appears to
inhibit the expression of IL-2, thereby suppressing the development of an immune
response [183].
PPARδ is expressed in myeloid cells and its expression is induced during
differentiation of human macrophages. Upon activation by its ligands, this receptor can
potentially increase triglyceride accumulation in macrophages [184]. PPARβ/ has also
been reported to regulate the production of TNF-α and IFN-γ and is thereby suggested to
be involved in wound healing [172].
42
6. Present investigations
The work presented in this thesis was designed to characterize how the murine
immune system is influenced by exposure to PFOA and PFOS.
6.1 Specific aims
Paper I. To examine the effects of 0.02 % (w/w) PFOA or PFOS in the diet on the
innate immune system of male C57BL/6 mice.
Paper II. To characterize the effects of 10-day dietary treatment with different doses
(1–0.001 %, w/w) of PFOS on the adaptive immune system of mice and
also to examine the possible involvement of PPAR in these phenomena.
Paper III. To determine whether sub-chronic (28-day) dietary exposure to an
environmentally relevant dose of PFOS (250 g/kg body weight per day)
is immunotoxic toward male B6C3F1 mice.
Paper IV. To develop a simple and more effective procedure for the isolation of
intrahepatic immune cells from mouse liver based on mechanical
disruption.
Paper V. To characterize the effects of 0.002 % PFOA or 0.005 % PFOS (sub-
immunotoxic doses) on the histology and immune status of the murine
liver.
43
7. Methodological considerations
The Materials and Methods employed in Papers I-V are described in detail in the
attached articles themselves. Below some key aspects of the methodology are considered.
7.1 Animals
Throughout our study we used the mouse as our experimental animal, because of
the paradigmatic use of this rodent species in immunological research. The murine
immune system is quite similar to that of humans and, therefore, the reaction of mice to
environmental factors can provide valuable information concerning potential
immunotoxic risk to humans. Moreover, mice are small, inexpensive, and easily
maintained and reproduce rapidly. The mouse strains we used here were; C57Bl/6,
129/Sv (wild type and PPAR knock-out) and B6C3F1. (Papers I-V)
7.2 High performance liquid chromatography- mass spectroscopy-mass
spectroscopy (HPLC-MS-MS)
Our collaborators at the 3M Company (St Paul, Minnesota, USA) have developed
a highly sensitive, selective and reproducible HPLC-MS-MS procedure for determination
of PFOA and PFOS in complex chemical mixtures. (Papers I, III and V)
7.3 Flowcytometry
This technique enables reliable identification and quantification of small numbers
of cells (1 in 10000) of a given phenotype among large mixed populations. (Papers I-V)
7.4 In vitro cell culture and stimulation
The cultures of peritoneal, bone marrow, spleen and liver-derived cells were
established to measure the production of various cytokines and antibodies in RPMI-1640
or D-MEM. (Papers I, III-V)
RPMI-1640 contains a bicarbonate buffering system, together with amino acids
and vitamins, and is commonly employed to support the growth of both normal and
neoplastic leukocytes.
44
7.5 Assay of cell proliferation
The MTT (methylthiazol tetrazolium) assay is based on the conversion of the
yellow tetrazolium salt-MTT to purple-formazan crystals by mitochondria and is
routinely used as a quantitative indicator of metabolically active cells. (Paper IV)
7.6 Propidium Iodide (PI) and the Annexin-V assay
PI staining solution is routinely used to assess plasma membrane (PM) integrity in
the Annexin V assay. PI stains DNA, in cells that have lost their PM integrity and
become permeable to PI as a consequence of either apoptosis or necrosis. (Paper IV)
45
8. Results
Paper I: High-dose, short-term exposure of mice to perfluorooctanesulfonate
(PFOS) or perfluorooctanoate (PFOA) affects the number of circulating neutrophils
differently, but enhances the inflammatory responses of macrophages to
lipopolysaccharide (LPS) in a similar fashion
After characterizing the effects of high-dose, short-term dietary exposure of mice
to PFOS or PFOA on adaptive immunity, we examined a number of parameters
associated with the innate immune system of mice, including activation by bacterial LPS
after dietary treatment with 0.02 % PFOS or PFOA for 10 days.
A first indicator of possible effects on the innate immune system, were the
reductions in the total numbers of circulating WBC (leukopenia) and lymphocytes
(lymphopenia), following exposure to either compound, whereas the number of
circulating neutrophils was decreased only by PFOA. Neutrophils are key cellular
mediators of innate immune responses and these findings thus provide an initial
indication that at least PFOA may exert an adverse effect on cells in the bone marrow.
The total numbers and relative proportions (percentage) of macrophages in three
immune compartments, i.e., the peritoneal cavity, bone marrow and spleen, are
influenced differently by exposure to PFOA or PFOS. Both of these perfluorochemicals
elevated the proportion, but not the total number of macrophages (CD11b+) among the
cells isolated from the peritoneal cavity; reduced both the proportion and total number of
these cells in the bone marrow; but had no significant effect on the size of the splenic
population of macrophages, despite the fact that the overall cellularity of the spleen was
strikingly reduced. Thus, it appears possible that bone marrow macrophages are
susceptible to, e.g., induction of necrosis and/or apoptosis by PFOA or PFOS, whereas
the resident macrophages in the peritoneal cavity and spleen are resistant.
To examine the possible influence of PFOS and/or PFOA on the functional
properties of peritoneal, bone marrow and splenic macrophages, we determined the
ability of these cells to produce TNF-α and IL-6 ex vivo with and without stimulation by
LPS (100 ng/ml). In the absence of this potent activator of macrophages, peritoneal cells
isolated from mice treated with either compound produced significantly enhanced levels
46
of both of these cytokines. The bone marrow cells from animals administered PFOA
secreted elevated levels of TNF-α whereas the production of this latter cytokine by spleen
cells is reduced by both PFOS and PFOA. In vivo exposure to PFOS potently enhances
the production of both TNF-α and IL-6 by peritoneal and bone marrow cells after in vitro
LPS stimulation while, at the same time, attenuating the corresponding production of
TNF-α by spleen cells.
A similar pattern is observed following treatment with PFOA, except that in this
case, the ex vivo production of TNF-α and IL-6 by spleen cells in response to in vitro
stimulation with LPS was also significantly enhanced. Since macrophages are the major
producers of these cytokines in these three compartments, we conclude that exposure to
either PFOS or PFOA alters the functionality of these cells. Together, these findings
suggest that PFOS and PFOA may sensitize macrophages to external insults, in particular
by infectious agents.
The observations documented above raise the question as to whether exposure to
PFOS and/or PFOA renders mice more susceptible to inflammatory responses associated
with infection. In essential agreement with our in vitro findings, pretreatment with PFOS
or PFOA prior to the challenge with LPS further potentiates production of these cytokines
by both peritoneal and bone marrow cells, while attenuating the ex vivo responses of
splenocytes to LPS-induced inflammation. Potentiation of the production of TNF-α and
IL-6 in response to LPS by pretreatment with PFOS or PFOA suggests that these
compounds might render the animal more susceptible to disease conditions
(e.g., systemic autoimmune diseases) in which TNF-α and IL-6 play a pivotal role
[185,186].
Finally, we found that dietary exposure of mice to a 20-fold lower dose
(i.e., 0.001 %, w/w) of PFOS or PFOA for 10 days did not alter any of the parameters
examined here.
47
Paper II: The atrophy and changes in the cellular compositions of the thymus and
spleen observed in mice subjected to short-term exposure to perfluoro-
octanesulfonate are high-dose phenomena mediated in part by peroxisome
proliferator-activated receptor-alpha (PPAR)
Here, we characterized the effects of 10-day treatment with different dietary doses
(1–0.001 %, w/w) of PFOS on the immune system of male C57BL/6 mice and also
examined the potential involvement of PPAR in these phenomena.
First, the general toxicity of PFOS was determined by monitoring changes in body
weight and other signs of toxicity (e.g., ruffled fur, lethargy, poor grooming or other
changes in behavior). Mice receiving dietary concentrations higher than 0.02 % exhibited
a pronounced dose- and time-dependent weight loss, which was accompanied by lethargy
and poor grooming. The 0.02 % dose did not cause toxicity with respect to behavior or
appearance, although a significant reduction in body weight, food consumption, and the
weights of the thymus, spleen and epididymal fat pads occurred. In contrast,
hepatomegaly was observed at all doses tested. These findings indicate a relatively steep
dose–response curve for the other effects monitored here in mice and clearly reveal that
liver hypertrophy (hepatomegaly) is the most sensitive endpoint in this context.
To evaluate these phenomena in greater detail, we determined the effects of PFOS
at dietary levels which do not induce toxicity (i.e., 0.001, 0.005 and 0.02 %) on the
overall cellularity and phenotypically distinct cell populations of the thymus and spleen.
The highest of these doses led to a marked reduction in the total numbers of thymocytes
and splenocytes (84 % and 43 %, respectively), as well as in all of the individual
subpopulations of thymocytes and splenocytes.
Consistent with the results obtained by flow cytometry, the histology of the
thymus of mice treated with 0.02 % PFOS differed significantly from that of the control
animals. The thymic cortex of exposed animals was smaller and virtually devoid of cells
and, moreover, the cortical/medullary junction was not distinguishable, in contrast to the
well-organized junction in the control organ. At the same time, with the exception of a
moderate reduction in the size of the lymphoid nodules, the atrophy of the spleen caused
by this same dose of PFOA or PFOS was not associated with any other obvious structural
changes.
48
Since we have previously found that PPARα plays an important role in PFOA-
induced modulation of the murine immune system [148] and both PFOA and PFOS can
act as agonists for PPARα [187], we next compared the effects of PFOS on wild-type and
PPARα-null 129/Sv mice. Although there was a significant increase in liver weight and a
marked reduction in the weight of the spleen in both the wild-type and null animal, the
weights of the thymus and epididymal fat pads were reduced in the wild-type mice only.
As in C57Bl/6 mice, the total numbers of thymocytes and splenocytes, as well as all
subpopulations of thymocytes (33–73 %) and splenocytes (37–41 %) were reduced in the
wild-type 129/Sv mice. However, in the PPARα-null animals, the reduction in the total
numbers of thymocytes and subpopulations thereof was partially attenuated and the
effects on splenocytes largely, if not totally, eliminated. Thus this receptor plays a
significant role in the immunotoxic effects of high doses of PFOS.
Paper III
28-Day dietary exposure of mice to a low total dose (7 mg/kg) of perfluoro-
octanesulfonate (PFOS) alters neither the cellular compositions of the thymus and
spleen nor humoral immune responses: Does the route of administration play a
pivotal role in PFOS-induced immunotoxicity?
A recent study had concluded that sub-chronic (28-day) exposure of mice by oral
gavage to doses of PFOS that result in serum levels comparable to those found in general
human populations suppress adaptive immunity [150]. Because of the potential impact of
these findings on environmental research and monitoring, we examined whether sub-
chronic dietary exposure (a major route of human exposure) to a similarly low-dose of
PFOS (250 g/kg body weight/day) also suppresses adaptive immune responses in male
B6C3F1 mice.
At the end of the treatment period, the exposed mice had gained somewhat less
weight than the untreated controls and the liver mass was elevated significantly; whereas
the masses of the thymus, spleen and epididymal fat pads were not significantly altered.
This dietary exposure had no effect on the total numbers of circulating leukocytes or of
cells in the thymus and spleen. Nor did this treatment alter the relative numbers of the
phenotypically distinct types of cells present in the thymus (i.e., immature CD4−/CD8−
49
and CD4+/CD8+ lymphocytes, mature Th cells and CTL, NKT and NK cells) or spleen
(i.e., B lymphocytes, Th cells and CTL, γδT cells, NKT and NK cells, macrophages,
granulocytes and plasma cells).
In order to determine whether such 28-day low-dose exposure to PFOS affects
humoral antibody responses to a T cell-dependent or T cell-independent antigen, PFOS-
treated and control mice were immunized with sheep red blood cell (SRBC) or TNP-LPS
on day 23 of the treatment period and antibody responses to this antigen monitored 5 days
later. First, we found no difference in the proportion of plasma (CD138+) cells in the
spleen, or in the number of these cells that were secreting anti-SRBC IgM antibodies
(employing flow cytometry and the plaque-forming assay), respectively. To be even more
certain of these findings, we employed ELISA procedures to measure circulating levels of
both IgM and IgG antibodies directed specifically against SRBC and TNP and, once
again, found no differences between the two groups. From all of the above findings we
conclude that a 28-day exposure of adult male B6C3F1 mice to dietary levels of PFOS
that results in serum concentrations (11.6 g/ml) one or two orders of magnitude higher
than those observed in occupationally exposed workers, does not compromise the
adaptive immune system and, perhaps, the route of administration may have a
considerable impact on the immunotoxic effects exerted by PFOS.
Paper IV
Isolation of murine intrahepatic immune cells employing a modified procedure for
mechanical disruption and functional characterization of the B, T and natural killer
T cells obtained
Intrahepatic immune cells (IHIC) are known to play central roles in
immunological responses mediated by the liver (see the Introduction above) and isolation
and phenotypic characterization of these cells is of considerable importance. Therefore,
we developed a simple procedure for the mechanical disruption of mouse liver that allows
efficient isolation and phenotypic characterization of IHIC. These cells were compared
with the corresponding cells purified from the liver after enzymatic digestion with
different concentrations of collagenase and DNase.
50
The mechanical disruption resulted in appreciably higher cell yields (64 %) than
those obtained by enzymatic digestion (50 %). Phenotypic characterization of the purified
IHIC by immunofluorescence staining and flow cytometry revealed that the IHIC isolated
following our mechanical disruption were, as expected, heterogeneous in composition,
containing both innate and adaptive immune cells of which B, T, NK and NKT cells,
granulocytes, macrophages, γδT and so-called myeloid suppressor cells, were the major
populations. The composition of IHIC obtained with enzymatic digestion was
significantly different, containing a higher percentage of B cells but, at the same time,
considerably lower proportions of NKT and γδT cells. These findings indicate that
murine intrahepatic NKT cells are highly sensitive to treatment with collagenase and
DNase.
In order to evaluate whether IHIC obtained with our improved procedure for
mechanical disruption were functionally intact, we examined in vitro the proliferative
responses of the B, T and NKT cells, as well as the production of IgM by B cells and
IFN-γ by T cells and NKT cells upon exposure to their specific activating stimuli. In
response to LPS, hepatic B cells proliferated and produced significant amounts of IgM;
T cells responded to ConA by producing high levels of IFN-γ; and NKT cells responded
to α-GalCer by synthesizing significant amounts of IFN-γ. These observations
demonstrate that these three cell populations in IHIC isolated following the improved
mechanical disruption are functionally immunocompetent.
Paper V
Dietary exposure to perfluorooctanoate or perfluorooctane sulfonate induces
hypertrophy in centrilobular hepatocytes and alters the hepatic immune status in
mice
As shown above, as well as by numerous other studies, the liver is the major
organ affected by the exposure of rodents to PFOA or PFOS. The hepatic effects of these
compounds include pronounced hepatomegaly, with long-term exposure elevating the
incidence of liver tumors in the rat [132]. Accordingly, we investigated the effects of
these compounds on liver structure and the hepatic immune system.
51
Dietary treatment of male C57Bl/6 mice with 0.002 % PFOA and 0.005 % PFOS
for 10 days exerts minimal or no effect on food intake, body weight gain, the mass of the
thymus, spleen and fat pads, serum levels of alanine aminotransferase (ALT) or aspartate
animotransferase (AST), hematocrit, or blood level of hemoglobin. At the same time,
PFOA- and PFOS-exposed animals exhibited a significant increase in liver mass
(1.6- and 1.4-fold respectively); with a moderate increase in the serum activity of alkaline
phosphate (ALP) (1.4-fold) and a reduction in the serum level of cholesterol (by 24 %)
and triglyceride (25 %).
Microscopic examination of liver sections exhibited structural alterations,
particularly in the parenchymal cells. With both compounds, the hepatocytes surrounding
the central vein were hypertrophic and displayed cytoplasmic acidophilic granules.
PFOA was found to induce a pronounced elevation (200 %) in the total numbers
of non-parenchymal IHIC. Moreover, the total numbers of phenotypically distinct types
of myeloid-, lymphoid- and erythroid-related cells (granulocytes, myeloid suppressor
cells, macrophages, Th cells, CTL, NK cells, T and erythroid progenitor cells) were
elevated. In contrast, exposure to PFOS enhanced only the numbers of erythroid
progenitor cells, without affecting other types of IHIC. Following exposure to PFOA or
PFOS, hepatic levels of the cytokines TNF-, IFN- and IL-4 were lower than in the
livers of control animals. Finally we found that exposure to PFOA or PFOS induced
4 to 5-fold increases in hepatic levels of erythropoietin, together with elevating the
numbers of erythriod progenitor cells present. Therefore we could conclude that the
alterations in the structure and functions of parenchymal and non-parenchymal cells in
the liver upon exposure to PFOA or PFOS might alter the susceptibility of this organ to
other toxins, drugs and hepatotropic infectious agents.
52
9. Concluding remarks
The five major findings documented here are the following:
I. High-dose, short-term exposure to either PFOS or PFOA activates certain
components of innate immunity in mice, in contrast to their suppressive
effects on adaptive immunity.
II. Short-term immunomodulation caused by PFOS in mice is a high-dose
phenomenon, with the thymic changes being partially dependent on the
PPAR, and splenic responses largely eliminated in its absence. On the other
hand, hepatomegaly is independent of PPAR.
III. Sub-chronic dietary exposure of mice to PFOS for 28 days, resulting in serum
levels approximately 8–85-fold greater than those observed in occupationally
exposed individuals, does not exert adverse effects on adaptive immunity.
IV. The simple procedure for the mechanical disruption of mouse liver described
here results in more efficient isolation of functionally competent IHIC for
various types of investigations.
V. In mice, PFOA- and PFOS-induced hepatomegaly is associated with
significant alterations in hepatic histophysiology and immune status, as well as
induction of hepatic erythropoiesis.
53
10. General discussion and future perspectives
Upper-bound estimates of serum concentrations in general human populations are
100 and 14 ng/mL for PFOS and PFOA, respectively [188,189]. Because PFOS and
PFOA are persistent in the environment [100,190] have long elimination half-lives in
humans [191,192], and have been detected in most serum/plasma samples from the
general population [188,192,193] it is important to consider the potential relevance of
immune changes observed here in mice to the body burden of PFOS and PFOA in the
general population. We observed that changes in the immune system occurred only at a
dose (0.02 % in the diet) that produced significant reductions in body weight and fat.
Because PFOS and PFOA are not metabolized [194], are distributed primarily in the
extracellular space [191], and are eliminated slowly in humans [192], serum
concentrations can be used as a measure of the body burden [100,193]. These properties
allow bridging of differences in the elimination rate between species in connection with
comparison of body burdens. As a result, serum concentrations associated with effects
and no-effect in experimental toxicological studies on rodents can be compared to those
measured in human biomonitoring studies, as a means of evaluating potential health risk.
The findings in Paper I, demonstrate that at least certain perfluorinated
compounds can exert adverse effects on the innate branch of the immune system, an
important defence system found in all plants and animals. The mechanisms underlying
the immunotoxic effects of both PFOS and PFOA on both the adaptive and innate
branches of the immune system require further elucidation. Toxicokinetic investigations
will help clarify whether these compounds exert their immunotoxic effects directly
(e.g., via PPAR or PPAR activation in primary and/or secondary immune organs) or
indirectly (e.g., by being accumulated in the same organs). Investigations of this nature
are currently being performed in our laboratory.
We have recently observed that the bone marrow, a primary immune organ, is a
major site for accumulation of PFOS in the mouse [195]. Our subsequent investigation on
the effects of PFOA and PFOS on hematopoietic cells in the bone marrow of mice
revealed that while exposure to low doses of these compounds affects mainly the
development of B lymphocytes, dietary administration of high doses exerts adverse
effects on the cells of most (if not all) hematopoietic lineages including the lymphoid,
54
myeloid and erythroid lineages. Since PPAR apparently is not expressed in murine bone
marrow cells [196], PFOA and PFOS may interfere with hematopoiesis via a mechanism
independent of this receptor. We hypothesize that these perfluorinated compounds hinder
the development of immune cells in the BM by disrupting cell-to-cell communication in
this organ, which in turn reduces the production of B cells and T cell progenitors. We
believe that testing this hypothesis would provide insight into the mechanisms underlying
the immunosuppression caused by PFOA and PFOS.
Moreover, Bogdanska and coworkers [195] have reported that PFOS accumulates
in the fetal liver, where hematopoiesis occurs prior to birth and, in addition, after birth the
pups looked pale, probably because the supply of oxygen was insufficient [80]. It would
be extremely interesting to examine the generation of fetal immune cells following
treatment of the pregnant dam with these fluorochemicals.
The dietary exposure employed in Paper III would appear to be a more realistic
model for human exposure than administration by gavage. At the same time, it should be
emphasized that humans and other organisms are exposed to PFOS (and PFOA) for much
longer periods than 28 or even 60 days, elimination half-lives being 5.4 and
3.8, respectively. In addition, there may be certain subpopulations of humans and other
animals that are particularly susceptible to the immunotoxic effects of this
fluorochemical. Obviously, further studies on these issues are required, e.g., exposure of
the mice at an environmental dose of PFOA or PFOS for 6 months or 1 year, with
subsequent evaluation of their immune status.
In Paper IV, we have developed a simple procedure for mechanical disruption of
mouse liver that allows isolation of IHIC in yields considerably higher than those
achieved by other reported procedures. In addition, the cells thus isolated are amenable to
culturing and are functionally immunocompetent. This procedure should prove to be
highly valuable in connection with attempts to elucidate the hepatic mechanisms
underlying the development of immunological tolerance, as well as other immunological
responses mediated by the liver. Furthermore, it will be of considerable interest to
examine changes in the composition and activities of IHIC in response to environmental
agents such as pathogens and xenobiotics.
55
Our findings in Paper V demonstrate that exposure of mice to 0.002 % PFOA or
0.005 % PFOS induces pronounced hypertrophy in centrilobular hepatocytes and alters
the hepatic immune status in mice. Further studies should be designed to determine the
consequences of the hepatic immune alterations caused by PFOA or PFOS, as well as to
elucidate whether these compounds exert their effects directly (e.g., by binding to
PPARα, γ and/or β or the erythropoietin receptor) or indirectly (e.g., by inducing hypoxia
in the cells affected, thereby stimulating erythropoietin production). Investigations of this
nature are currently being performed in our laboratory.
Finally, detailed immune testing could be performed on occupationally or
environmentally exposed human populations e.g., those living in close proximity to
fluorochemical manufacturing plants. Although it is now well established that PFOA and
PFOS exert suppressive effects on the adaptive immune system in rodents [146,150,197]
little or no information is available on the possible influences of these compounds on the
human immune system. Evaluation of this issue is of considerable importance in in vitro
studies, designed to test for alterations in the functions of both un-stimulated and
mitogen-stimulated peripheral blood mononuclear cells (PBMCs) in response to PFOA or
PFOS might provide valuable information concerning the effects of these compounds on
the human immune system. Furthermore, the specific humoral immune responses
following vaccination (e.g., for influenza virus) of occupationally-exposed individuals
could be compared to the responses of un-exposed individuals. Finally, elucidating the
effects of perfluorinated chemicals in mice reconstituted with human immune cells, so-
called SCID-human mouse, might also be highly valuable [198].
56
11. Acknowledgements
My thesis has finally been completed after invaluable contributions in various
ways by several people and dedicated researchers. Especially, I would like to
acknowledge:
Joe DePierre, my supervisor, for your excellent scientific guidance and never-
failing enthusiasm and support during the last 6 years. Thanks also for always taking the
time for inspiring discussions and for giving constructive criticism and making the work
stimulating and joyful. We enjoy your songs and the delicious food at your place.
Barbara, thanks for your care and your company at the gym.
Manuchehr Abedi-Valugardi, my hands-on advisor, for enthusiastically
introducing me into this department. Thanks also for stimulating discussions, planning
and for always seeing the bright side of a problem. Your tremendous support and caring
have given me strength and confidence throughout the years. I have really enjoyed your
positive attitude, which has made the work more fun.
Other members of our group: Buck Nelson for scientific advice and critical
reviewing our manuscripts and your intelligent and insightful advice at our weekly group
meetings; Stefan Nobel, for your support, encouragement and correction of the Swedish
abstract; and Jasna Bogdanska, for all the nice hours we spent together in the lab, for your
care and for being such a very nice colleague, and for our enjoyable discussions during
the morning coffee break. All of my other co-authors for their important contributions to
the different studies.
Stefan Nordlund, for generous support and care during my entire PhD period.
Thanks for your valuable comments on my thesis. Thanks to Inger Carlberg for being
available to discuss any and all issues in a caring fashion.
Elzbieta Glaser and Åke Wieslander for your intelligent advice and dignified
personalities.
John Butenhoff, from the 3M Company, for making this collaboration possible.
Thank you for your time, active interest in our work, detailed comments on our
manuscripts and valuable suggestions concerning the thesis. Thanks to Dave J Ehresman
for analyzing the serum samples.
57
My dear colleagues at the department: Anki, Ann, Haidi, Maria, Lotta and Lollo
for your patient help with administrative things and ´´always-finding-a-solution``;
Torbjörn and Peter, for helping me with computer problems; Håkan and Bengt for
technical support. Special thanks to Bogos, for sharing your knowledge, for delivering
packages and for your friendly manner. You were the first person who helped me find
Joe´s office when I was almost lost in this big department. I wish you could have been
here to share my joy about completing my PhD thesis.
Eva, Solveig and Ellinor in the animal house, for your kind help in taking care of
the mice. Eva, you create a pleasant atmosphere in the animal house and always welcome
me there with smile.
The great atmosphere at DBB is created by wonderful people: Candan, Pedro,
Tiago, Beata, Catarina, Pilar, Gabriela, Joachim and Martin Ott group for the good times
and enjoyable moments. You are always smiling and are nice to talk to. Thank for our
non-scientific chats during the lunch and coffee breaks. Salomé, the friendly scientist,
your energy will guarantee you a bright future in science. Changron, for helping with the
practical aspects of this thesis. I am grateful to both of you for taking the time to help me
prepare for the ”big exam”.
My degree project and summer students, you each made tremendous progress
during your stay in our lab. I learned so much from you. You have bright futures ahead.
I also wish to express my sincere gratitude to all the people in Lappis, my good neighbors
Susmita, Shapla, Rekha and your families. I will never forget the pleasant times we spent
together, especially the BBQs. Thank you for friendship and for being such a nice group
of people. I am grateful to Rekha and Emon for your help with the final preparations
required to get this thesis into print. Thanks and good luck with your own PhDs.
Zarina Nahar Kabir, my cousin, we are lucky to have you. Thanks for your care
and support. Tonima and Shanta, my niece, for your kind heart and dignified personality.
Dr. Atiqul Islam for your support and great sense of humor.
Tamanna, my friend, we had delighted times together when you were in Stockholm.
Khaleda, my sister, thank you for always being there for me. Many thanks for all
of your kind help and support at the beginning when I didn’t know where any place in
Stockholm was. Your enthusiasm for science has made a lasting impact on me. Your
58
encouragement has been important. Best wishes in your research and life. Apu, my
brother-in-law, for helping to create a stimulating and friendly environment outside the
world of science. Thank you for your great laughter. My cutypie nephew, Arian, I feel
delightful when you are around. I like your attitude and smile.
My mother, for your never-ending love, raising me with your spirit and
encouraging me to reach this level. To my father, I can never find a more trustworthy,
lovely and charming person than you in all my life. This thesis is as much yours, mother
and father, as it is mine. My brother (and his family), I appreciate so dearly your constant
encouragement. Thanks for supporting our parents in this critical situation.
I thank my parents-in-law for their affection and constant support.
My dearest husband, Jubayer, for always putting my needs ahead of your own,
and for taking care of everything when I am busy. Thanks a lot for editing my thesis and
for the invaluable comments.
Above all, my lovely little daughter, Ilma, for being the joy and the meaning of
my life. For your big smile and your patience during these years and especially during the
final work on this thesis. You have such a nice personality.
These studies were made possible by unrestricted grants from the 3M Company
(St Paul, Minnesota, USA).
59
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