Technical Report 122

Relevance of alveolar macrophages

Alveolar macrophages (AM) represent primary phagocytes of the innate immune system and are of fundamental importance in mediating the removal of inhaled particles from the lung. Phagocytic and cytotoxic activity of AMs along with mucociliary transport provides an effective, nonspecific pulmonary defence. Consequently a rapid increase of phagocytic active cells like polymorphonuclear leucocytes (PMNs), neutrophils and AMs is observed in the lung following particulate exposure. In fact, AM accumulation can be considered an early indicator of pulmonary particle deposition. Several studies have shown that AMs can be differentiated based on their anatomical location and biological activities. They consist of several functionally distinct subpopulations which differ from each other by their phagocytic activity as well as their biochemical, cytotoxic/cytostatic cellular functions, and in their ability to synthesise and release soluble mediators. Species differences in composition, localisation and function between the different AM subsets may therefore account for observed differences in responses to inhaled particulates. Depending on their localisation, intravascular, interstitial, pleural and surface associated AMs can be distinguished. Because surface macrophages participate primarily in innate immune responses like phagocytosis, differences in this subpopulation may be responsible for differences in clearance rates of particulates. Surface macrophages can easily be obtained by bronchoalveolar lavage (BAL). Rodent BAL cells consist of ≥95% macrophages, few lymphocytes and neutrophils whereas human BAL cells normally consist of ≥80% macrophages, about 18% lymphocytes and few neutrophils. This is in line with findings that the shortest macrophage clearance times are reported for rodents where deposited micrometre-sized particles remain on the epithelial surface of the lung (Lehnert et al, 1989; Ellender et al, 1992). Longer clearance times are found in humans, monkeys, dogs and guinea pigs (Snipes 1989; Kreyling 1990).

Based on reported retention kinetics of poorly soluble particles, generic elimination half-times of 60 days for rats and approximately 1 year for humans can be assumed (Oberdörster 1995). According to Pauluhn (2011), the roughly 7-fold greater AM pool of humans relative to rats results in a proportionally longer clearance of approximately 420 days making the anticipated clearance half-time in humans plausible. Whereas the alveolar clearance rate in humans seems to be independent on the particle load, the clearance rate in rats depends on the amount of particles in the alveolar region which may contribute to a more pronounced impairment of macrophage mediated alveolar clearance and thus the higher susceptibility of rats to lung overload (Brown et al, 2005).

Marked species differences exist also in the cell size of alveolar macrophages with AMs from humans being significantly larger than those from rats, hamster or monkeys. Based hereupon, number and size range of phagocytised particles may also differ among species (Krombach et al, 1997).

Beside phagocytosis, macrophages are also one of the most active secretory cell types releasing a multitude of mediators. In this respect they are involved in the regulation of nearly all aspects of host defence, inflammation, wound healing and homeostasis (Murray and Wynn 2011, Laskin et al, 2011). This diverse biological activity of macrophages is mediated by two phenotypically distinct subpopulations of macrophages, classically activated "M1 macrophages” and "alternatively activated "M2 macrophages”. Classically activated macrophages (M1) are produced during cell-mediated immune responses and exhibit microbicidal and tumouricidal activity. By releasing reactive oxygen/nitrogen species (ROS/RNS) and pro-inflammatory cytokines (e.g. IL-1, IL-6, IL-12, IL-23, TNF-α), they also exert strong anti-proliferative and cytotoxic activities. Although M1 macrophages are vital components of host defence, their activation must be tightly controlled because the produced cytokines and mediators can promote host-tissue damage. In contrast to pro-inflammatory "M1” macrophages, alternatively activated "M2” macrophages exhibit strong anti-inflammatory activity. Consequently, M2 macrophages play an important role in wound healing. This activity is mainly related to the release of cytokines, bioactive lipids and growth factors like TGF-ß. However, similar to classically activated pro-inflammatory M1 macrophages, anti-inflammatory M2 macrophages can also be detrimental to the host when dysregulated. Excessive release of mediators and growth factors then can lead to pathologic fibrogenic responses (Mosser and Edwards, 2008).

Based on all the available experimental data it can be concluded that AMs respond to exposure against toxicants normally through a carefully balanced system consisting of "M1”macrophages which release pro-inflammatory and cytotoxic mediators important in host-defence and "M2” macrophages which are involved in down-regulating inflammatory processes and the initiation of wound-repair.

However, prolonged exposure to high levels of particles may provoke an imbalance resulting in hyper-responsive AMs followed by the dysregulated release of mediators that promote acute tissue injury and the progression of chronic diseases like fibrosis and eventually cancer; in the case of the rat. Differences in the quantitative activation of M1 or M2 macrophages as well as qualitative functional changes of AM may thereby account for the observed species differences in pulmonary responses following particle induced lung overload. Following such exposures, AMs with an inflammatory phenotype ("M1” macrophages) release pro-inflammatory cytokines as well as reactive oxygen (ROS) and reactive nitrogen (RNS) species that can be severely damaging to surrounding cells and tissue and may initiate abnormal inflammatory reactions.

ROS and RNS, e.g. superoxide anion, hydrogenperoxide, hydroxyl radical, nitric oxide, peroxynitrite, are generated via enzyme-catalysed reactions. Produced in large quantities by classically activated macrophages during inflammatory pulmonary responses, the resulting oxidative and nitrosative stress can lead to severe tissue injury. There are ongoing discussions that species differences in the oxidative capacity of AMs may be important for the different observed pulmonary responses to particulates. Data from Dörger et al (1997a) suggest that such species differences exist. The oxidative capacity was about 5-fold higher in rat AM than in hamster AM. Whereas the oxidative capacity of hamster AM appeared to be based mainly on the formation of ROS, the authors suggested that rat AM possess an additional oxidative system. In this respect an observation made by Schneemann et al (1993) may be of interest that in contrast to rodent AMs, human mononuclear phagocytes lack nitric oxide synthetase. Additionally, Dörger et al (1997b) and Jesch et al (1997) also reported that nitric oxide formation could only be observed by rat AMs, but not in AMs from hamsters, monkeys or humans. The authors concluded that distinct regulatory mechanisms of the nitric oxide pathway in alveolar macrophages from these four different species seem to exist. Taking into account that nitric oxide rapidly reacts with superoxide anion to form the relatively long-lived strong cytotoxic oxidant peroxynitrite, the higher sensitivity of rats towards both, inflammation driven non-neoplastic and unique neoplastic responses may be plausible. When protonated, peroxynitrite will decompose into NO2, as well as nitrate. These species may then interact with each other, as well as with O2 or ROS to form higher oxides of nitrogen which may oxidise thiols and a variety of amino acids including methionine and cysteins. It therefore can be concluded that species differences in the formation of RNS, as revealed between 26 ECETOC TR No. 122
rats compared to other species, may contribute to the observed differences in pulmonary tissue injury following chronic exposure to high concentrations of poorly soluble particles.

In contrast to M1 macrophages, alternatively activated M2 macrophages exhibit strong anti-inflammatory activity; play an important role in wound healing and when hyper-responsive also in the development of fibrosis (Wynn & Barron 2010). This activity is mainly related to excessive release of cytokines, bioactive lipids and growth factors like TGF-ß, a known mediator of fibrosis by stimulating the production of extracellular matrix proteins (Pulichino et al, 2008). Species differences in the pulmonary micro-environmental conditions, responsible for the activation of "M1” and "M2” macrophages and/or differences in generating mediators as well as ROS/RNS, may therefore play an important role in some of the observed species differences in lung injury following chronic exposure to PSPs of low toxicity.