Particle Toxicology - Chapter 9 pps

The article will focus on the effects of a-quartz, asbestos, and environmental particulate air pollution particles and how these different particles activate epithelial cells and macroph

9 Proinflammatory Effects of

Particles on Macrophages and Epithelial Cells

Vicki Stone

School of Life Sciences, Napier University

Peter G Barlow

Queen’s Medical Research Institute, University of Edinburgh

Gary R Hutchison

Medical Research Council, Queens Medical Research Institute

David M Brown

School of Life Sciences, Napier University

CONTENTS

9.1 Particle-Induced Inflammation and Disease 183

9.2 The Role of Epithelial Cells in Driving Particle-Induced Inflammation 185

9.3 The Effects of Particles on Macrophage Signaling Mechanisms 187

9.4 Interactions Between Macrophages and Epithelial Cells 189

9.5 Using Macrophages and Epithelial Cells as a Model for Studying Particle-Induced Inflammation—Conclusion 191

References 191

This chapter aims to compare the ability of a number of potentially pathogenic particles in terms of their ability to induce inflammation and disease The article will focus on the effects of a-quartz, asbestos, and environmental particulate air pollution particles and how these different particles activate epithelial cells and macrophages leading to inflammation

9.1 PARTICLE-INDUCED INFLAMMATION AND DISEASE

As described in the previous chapter, inflammation is considered to play a key role in driving disease induced by a number of pathogenic respirable particles For example, carcinogenic and pro-fibrotic particles such as a-quartz and asbestos have both been shown to induce a chronic

response coupled with the surface, chemical, or physical reactivity of the particles is thought to

inflam-mation can result in a number of processes that contribute to the induction of fibrosis and carcinogenesis For example, the inflammatory process results in the production of mitogenic

183

stimuli that induce epithelial cell proliferation (Figure 9.1).5As cell proliferation rates increase, the chance of successful repair to damaged deoxyribonucleic acid (DNA) prior to cell division is diminished and hence the risk of passing on a mutation to daughter cells is enhanced In addition, such particles have been demonstrated to generate reactive oxygen species (ROS) due to activity at

lead to oxidative stress and damage to macromolecules such as DNA, again increasing the potential for mutations A more detailed discussion can be found in the Chapter on Genotoxicity of Particles (Schins & Hei)

Particulate air pollution particles, PM10(particulate matter collected through a size selective inlet with 50% efficiency for particles of 10 mm aerodynamic diameter), are associated with short-term

and variable with location and time, however a number of studies have suggested that various

Much of the information that is available relating to the toxicology of ultrafine particles (diameter less than 100 nm) has been obtained from the study of low toxicity, low-solubility

induce oxidative stress and inflammation increases Such results are likely to be relevant to the toxicology of engineered nanoparticles, although this may vary if the nanoparticles are soluble,

of macrophage phagocytosis and cellular uptake, or if the particles are extremely toxic (e.g., a-quartz) With the recent expansion of nanotechnology, engineered nanoparticles are used in a wide variety of applications including sunscreens, cosmetics, food, and medicine, as well as

Type II cell proliferation

PMN

Macrophage

Particle exposed Type I cell damage/death

DNA damage

Type I epithelial cell

Type II epithelial cell

+ROS Mitogens

Alveolus

Respiratory

bronchiole

FIGURE 9.1 A schematic diagram of the respiratory regions of the lung depicting the effects of particles on cell proliferation and mutation In the presence of pathogenic particles, reactive oxygen species (ROS) can damage the DNA of dividing cells leading to mutation The ROS can also activate intracellular factors that drive cell proliferation (AP-1) These factors combined with mitogenic factors released by the inflammatory cells (neutro-phils and macrophages) stimulate type II cell proliferation to replace the damaged type I cells An increase in cell proliferation rate increased the possibility that mutations are not corrected and hence become permanent

technical applications such as electronics This means that exposure to a wider range of nanopar-ticles is likely to occur for workers, consumers, and patients via a number of exposure routes including inhalation, ingestion, injection, and dermal adsorption Hence, the variety of target

between the surface area dose of the particle instilled into the rat lung and the ability to induce inflammation, as indicated by neutrophil influx 18 h after instillation Stoeger et al recently identified

a link between surface area and the ability of six different carbon particles to induce inflammation in

surface area, below which an acute proinflammatory responses could not be detected A similar study was reported by Tran et al using inhalation of TiO2and barium sulphate particles into the rat lung, but

tenfold difference in threshold could be due to animal size and species differences The subsequent translation of the threshold dose, identified by Tran et al into particle surface area per surface area of lung took into consideration that deposition of the particles would be most prevalent in the

assessment in relation to toxicological impact A simple explanation for the link between surface area and inflammation is that the surface/volume ratio changes dramatically at low radius and that the surface area is directly proportional to the number of atoms at the particle surface

Many papers have described the roles of macrophages and epithelial cells in driving the

will concentrate on the signaling mechanisms elicited in these two cell types on exposure to a variety of particles

9.2 THE ROLE OF EPITHELIAL CELLS IN DRIVING PARTICLE-INDUCED

INFLAMMATION

When inhaled particles deposit in the upper airways, they interact with epithelial cells that are ciliated and covered in thick, sticky mucus This allows the particles to be cleared by the wafting actions of the mucociliary escalator, causing the particles to be transported out of the lung airways

to be swallowed into the stomach, or to be blown from the nose In contrast, particles depositing in the respiratory parts of the lung, including the alveoli and terminal respiratory bronchioles, must be cleared by phagocytic cells such as macrophages In this region of the lung, the epithelium is not ciliated—instead the type I epithelial cells of the alveolus are large, thin, flat structures that are designed for gaseous exchange The type I epithelial cells are incapable of division due to their specialized nature If damaged by toxins or particles, the type I cells must be replaced by division of type II epithelial cells (type II cell hyperplasia) that subsequently differentiate into type I cells Type II cells are functionally very different to type I cells, with functions that include synthesis

type II cells are also capable of synthesizing a range of inflammatory mediators, so they play a key role in particle induced inflammation in the lung Particle deposition in the alveolus results in interaction with these epithelial cells, which generate chemotactic factors (e.g., IL8) to stimulate

of the clearance process A more detailed description of the chemotactic actions of epithelial products generated by particle treatment is provided below

Few studies have demonstrated an ability of ultrafine or nanoparticles to induce cytokine production by epithelial cells in vitro, unless studied at very high mass doses This is because the surface of the particles is very adsorbent, and hence binds the proteins released by the cells, resulting in an underestimation of the cytokine production Seagrave et al have also demonstrated

biologi-cally active, as it was able to induce neutrophil shape change We have recently found that this effect is not limited to cytokines such as IL8 and TNFa, but also includes the cytotoxicity marker

protein adsorption onto the particle surface in terms of the protein function and the particle toxicity requires further investigation; perceivable effects range from protein dysfunction to

Despite the lack of evidence that nanoparticles stimulate epithelial cells to generate cytokine proteins, there is, however, sufficient evidence to suggest that the messenger ribonucleic acid

The gene expression of many proinflammatory cytokines such as IL8 and TNFa is under the control of the transcription factor nuclear factor kappa B (NF-kB) NF-kB, when not activated, is retained in the cytoplasm of the cell by the binding of inhibitor kappa B (IkB) Phosphorylation of

with a-quartz led to a persistent depletion of IkB allowing proinflammatory signaling and IL8 expression to continue Treatment of A549 cells with asbestos has also been shown to induce DNA

were also found to induce NF-kB nuclear localization, DNA binding, and transcriptional activation

particles activate the expression of heat shock protein 70 (HSP70) by the A549 lung epithelial cell line HSP70 can prevent NF-kB activation by the stabilization of IkB kinase This means that HSP70 is a cytoprotective protein that exists in cells as a molecular chaperone, and during oxidative

HSP70 secretion has also been shown to be increased in response to pathogenic particles such

stimulated increased HSP70 secretion by A549 cells—an effect that was significantly inhibited by the addition of antioxidants, suggesting a role for ROS in the particle induced upregulation and release of this molecule The role of the released HSP70 is not fully understood, but extracellular HSP70 has been shown to activate macrophages leading to calcium influx, NF-kB activation and

associated with a proinflammatory effect, since release of HSP70 into the blood has been associated with a proinflammatory status We hypothesize that the differential activation of NF-kB and inhibition via HSP70 may be treatment dependent, with relatively low dose or low toxicity materials allowing HSP70 activation and upregulation of antioxidant defenses, while higher dose

or toxicity materials bypass this protective mechanism leading to NF-kB activation and

are not mutually exclusive and that there is either overlap or that they form part of a continuum Many of the cell signaling events described above include a role for oxidative stress or ROS For example, nanoparticle carbon black has been shown to deplete the antioxidant glutathione in the

tissue and bronchoalveolar lavage fluid glutathione content.52The pathology of asbestos has also

The role of ROS in driving the proinflammatory effects of many particle types is evidenced

by the ability of antioxidants to inhibit a variety of particle induced signaling events For example, as described above, nacystelyn prevented HSP70 nuclear localization in A549 cells

nacys-telyn, along with the other antioxidants such as curcumin, could prevent activation of the transcription factor NF-kB on exposure of A549 cells to pathogenic fibres Mannitol, a hydroxyl radical scavenger, has also been demonstrated to prevent nanoparticle carbon black induced

9.3 THE EFFECTS OF PARTICLES ON MACROPHAGE SIGNALING MECHANISMS

Not all particles entering the body are pathogenic; this is because macrophages play a major role in the clearance of foreign particles Alveolar macrophage make up approximately 5% of the total

Interstitial and intravascular macrophages are also present in the lung, but these are less well studied due to their inaccessibility by bronchoalveolar lavage

In order for macrophages to respond to a chemotactic stimulus, to migrate, to phagocytose a particle, and then to clear that particle from the tissue, a complex interaction of extracellular and

Figure 9.2) However, these pathways are open to modulation by environmental factors and by the toxic effects of the particle An alteration in such signaling pathways can lead to decreased particle clearance and hence increased risk of inflammation and disease

Inflammation but antioxidant defences not upregulated

DEP

PM 10

NPCB

Asbestos

PM 10

α-Quartz

HSP70 expression+

nuclear localisation Nrf-2

activation activationARE

Prevention of inflammation plus upregulated antioxidants

HSP70 secretion NF-κB activation

AP1 activation inhibitionARE ROS

ROS

Epithelial cells

Epithelial cells

Low level

exposure

High level

exposure

FIGURE 9.2 Hypothetical mechanisms involving epithelial cells by which particles may differentially regulate proinflammatory and antioxidant defense mechanisms PM10(respirable particulate air pollution) and nanoparticle carbon black (NPCB) have all been demonstrated to activate HSP70 nuclear localization, while diesel exhaust particles (DEP) have also been shown to activate the antioxidant response element (ARE), allowing antioxidant upregulation and prevention of inflammation Conversely, asbestos, PM10

and a-quartz have all been shown to activate NF-kB leading to cytokine gene expression and inflam-mation The two pathways are not suggested to be mutually exclusive, but may be a continuum or overlap

For most particles, the site from which particles must be cleared is usually the respiratory

well documented that intravenous injection of a variety of particles, including nanoparticles, results

in their accumulation within the reticulo-endothelial cells including the Kupffer macrophage cells

translocate across the pulmonary barrier will be taken up by this system of tissue macrophages Indeed, the studies demonstrating particle translocation from the lung do exhibit accumulation in

exposure routes will also include ingestion as components of foods and injection as components of medicines A description of cellular uptake and translocation mechanisms for nanoparticles are described and discussed in the chapter by Rothen-Rutishauser et al

Many studies have demonstrated a key role for macrophages in driving the proinflammatory response to pathogenic particles Of these studies, a large proportion have been conducted in vitro using both primary cells and cell lines, so the results generated—although focused initially on the lung—are now relevant to any potentially exposed tissue type Since nanoparticles are known to activate macrophages in vitro (described below), it is likely that regardless of the tissue type, macrophages can be activated by nanoparticle exposure, but that clearance will not be fully effec-tive, leading to a proinflammatory status However, this response may not be a universal reaction to nanoparticles, since modification with agents such as PEG is known to prevent uptake by the

demon-strating how drug delivery particles such as liposomes and polystyrene beads can be modified to

to be very successful for this purpose Surfactants decrease the van der Waals forces that are responsible for particle aggregation, and actually increase the repulsive forces Polymer-coated particles are thought to avoid macrophage uptake by prevention of opsinization of the particle surface While this field of research is fairly well advanced for nanomedicine, this data requires examination in order to allow application to other types of nanoparticles

In vitro studies with macrophages have demonstrated that nanoparticle carbon black and PM10 activate the expression of proinflammatory mediators such as tumor necrosis factor alpha

via a mechanism involving ROS This would suggest that oxidative stress or ROS are important in both the epithelial cell (as described above) and macrophage responses to pathogenic particles The signals induced then activate transcription factors such as NF-kB, leading to the subsequent

To date, most of the studies relating to the induction of inflammation and nanoparticles have concentrated on the upregulation of inflammation However, a number of signaling pathways are activated by oxidative stress that can protect the cell For example, activation of the antioxidant response element (ARE) by Nrf-2 leads to the upregulation of antioxidant defenses in response to ROS production ARE is a genetic sequence found in the promoter of many genes that controls the expression of antioxidant defense pathways, including enzymes such as heme-oxygenase-1 (HO-1)

proin-flammatory cytokines, while Nrf-2 activation leads to the induction of antioxidant defense

of diesel exhaust particulates induced the expression of HO-1 and GST in macrophages via a transcription factor Nrf-2 In other studies, the oxidant tert-butyl hydroperoxide and

actually contains a binding site for the transcription factor AP-1 We have previously demonstrated

binding to the ARE inhibited its activation thus preventing the upregulation of antioxidant defenses

This would suggest that in macrophages exposed to environmental particles or nanoparticles there is the potential for activation of pathways that both increase inflammation (via NF-kB and AP-1) and increase antioxidant defense mechanisms (via Nrf-2) Furthermore, these pathways have the potential to interact with AP-1 inhibiting ARE and therefore preventing upregulation of antioxidant defense mechanisms by Nrf-2

One of the primary roles of macrophages is to phagocytose foreign material Nanoparticle

uptake of the nanopartices has been associated with a subsequent decrease in the ability of the

suggest that particle clearance is not efficient in the presence of nanoparticles, allowing the particles

to persist in the lung, drive inflammation, and perhaps cross the epithelial barrier and gain access to the lung interstitium20and blood.57

However, macrophages obviously have a limited capacity to phagocytose particles of any type

In the future, it would be interesting to try to estimate the relative ability of macrophages to phagocytose particles of different sizes, and then to identify whether the maximum tolerated volume of particle uptake with particles of different size is comparable With the data generated thus far, it is difficult to determine whether the nanoparticles per se specifically inhibit the further uptake of larger particles, or simply “fill” or “overload” the cells preventing further particle uptake

The effects of asbestos on macrophage are also well studied The ability of asbestos to induce proinflammatory cytokine production by macrophages has been related to the fibre length, with

take up asbestos by phagocytosis Fibers of longer than 15 mm are not easily ingested and lead to a

into the cytoplasm of macrophages, although this could be as a consequence of cell death rather

asbestos fibres has also been shown to deplete glutathione and to activate NF-kB DNA, an effect

Particles of a-Quartz are thought to be highly cytotoxic to macrophages, such that uptake of a-quartz leads to inhibition of macrophage function, macrophage cell death, and subsequent

shown to induce calcium elevation in macrophages, but as a part of the cell death process rather

the macrophages, these particles must also be able to activate the cells leading to a proinflammatory response

9.4 INTERACTIONS BETWEEN MACROPHAGES AND EPITHELIAL CELLS

Of course, in the body, particles never encounter one cell type at a time, and instead the response is

a culmination of complex interactions between particles and many cell types There are a number of ways in which such interactions can be studied, including:

(i) Animal models

(ii) Lung slices or isolated organs

(iii) Co-cultures

(iv) Conditioned media from individual cell types and transferring these to other cells

in culture

With options (i)–(iv), attributing the inflammatory or signaling responses observed to any particular cell types can be difficult, but they have the advantage of providing a more physiologically relevant response

Our own studies using conditioned media suggest that exposure of epithelial cells to nanopar-ticle carbon black results in the generation of chemotactic factors that stimulate the migration of

macro-phages and that a complex interaction between macromacro-phages and epithelial cells plays a key role in driving the inflammatory response Once the macrophages migrate to and locate the nanoparticles,

it might be expected that these particles would be taken up by phagocytosis and subsequently cleared from the lung tissue by migration of the macrophage onto the mucociliary escalator, or by migration to the lymph nodes However, there may be a discord in the effectiveness of this mechanism when nanoparticles are involved Hypothetically, if particles can induce epithelial cells to secrete macrophage chemoattractants, a prolonged exposure of epithelial cells to nanopar-ticles may result in hypersecretion of chemoattractants into the alveolar space It is possible that this may disrupt the normal chemotactic gradient within the lung and result in particle-laden macro-phages remaining within the respiratory region instead of migrating to the mucociliary escalator for clearance The impact of nanoparticles on the chemotactic gradient within the lung clearly requires further investigation

adhesion molecules are important in vivo as they allow interaction with migrating leukocytes, facilitating their traffic through the tissue

Other methods of macrophage recruitment to sites of particle deposition could involve activation of the alternative complement cascade, resulting in the local generation of the

generation of macrophage chemotactic factors in blood serum Although serum is not generally present in the alveolar space, in times of prolonged inflammation, the lung vasculature may be compromised, allowing seepage of serum proteins into the alveolar space If nanoparticles were

to come into contact with these proteins, it is feasible that an excess generation of chemotactic substances could be generated, resulting in heightened inflammation This observation may also have ramifications with regard to particle translocation from the lung into the blood stream and systemic activation of complement could have serious effects on the cardiovascular system

the lung as particle-laden macrophages may be unable to migrate towards the mucociliary escalator

to be removed from the lung This would again result in prolonged macrophage retention and increased inflammation as a result

samples were found to activate proinflammatory (interleukin 1(IL-1B) and TNFa) and pro-fibrotic (TGFb) cytokine gene expression by macrophages, with those samples highest in metal content

proin-flammatory cytokines such as IL8, IL6, or TNFa by epithelial cells

and used to treat the macrophage cell line in order to ascertain whether these products would elicit changes in macrophage activity Despite the lack of IL8, IL6, or TNFa production measured in this experiment, the supernatant from these cells was very potent at inducing the expression of a number

of proinflammatory mediators (IL8 and granulocyte-macrophage colony-stimulating factor) by the

metal content was so potent that it inhibited macrophage cytokine production, probably due to toxicity These findings suggest cellular interactions are taking place, however the factors driving

these responses by epithelial cells are currently unknown Previous studies using conditioned media have highlighted the importance of cytokine/chemokine release and the role these molecules have

in driving effects both locally and systemically, particularly in the field of particle toxicology Schmidt et al published a study in which macrophages were treated with silica, a know agent of

found to modulate proliferation of fibroblasts, providing evidence that the particles induced release

of cytokines from one cell type to impact on the function of another cell type Albrecht et al also reported similar effects attributed to unknown factors in media created from lavage of quartz treated

inde-pendent, suggesting other mediators were involved in NF-kB activation and the associated inflammation Due to the complex overlapping and redundant nature of inflammatory signaling,

it is often difficult to attribute specific effects to one molecule; however, while studies using conditioned media are not always useful to determine exactly which factors are responsible for the cellular response, they are useful for providing information on the combined and overall effects

of the particle induced mediators

9.5 USING MACROPHAGES AND EPITHELIAL CELLS AS A MODEL

FOR STUDYING PARTICLE-INDUCED INFLAMMATION—CONCLUSION

As outlined above, a wide variety of studies indicate that different pathogenic particles induce oxidative stress and signaling mechanisms in both epithelial cells and macrophages that drive the inflammatory response leading to disease Many of the studies conducted to investigate the role of these two cell types in driving the inflammatory response have been conducted in vitro

In many cases, the in vitro cultured cells reflect closely the potency of the particles observed using in vivo models with respect to oxidative stress and the activation of proinflammatory signaling events

With the rapid expansion of nanotechnology and the potential for increased exposure to a wide variety of particles of unknown toxicity, it will be essential to exploit such in vitro protocols in order to prevent excessive animal testing It is therefore essential that a systematic assessment of the relevance and reliability of a number of in vitro models be conducted Such models are likely to include single cell types, cell lines, primary cells, and mixed cultures With such models, it is essential to prevent artifacts due to the use of particles (e.g., protein adsorption) Relevant controls (e.g., low-toxicity particles) must also be included, and of course, it will be necessary to compare the results to in vivo responses, using historical data where appropriate and possible Finally, the validation of such models will result in a valuable tool for the toxicity assessment of any potentially pathogenic particle in the future

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