Combustion-derived nanoparticles have been implicated in the adverse effects of ambient particulate air pollution (reviewed in ) In addition, the toxicological impact of nanoparticles has now come under increased scrutiny due to the emergence of a relatively new technological discipline; nanotoxicology. Studies have shown that nanoparticles can be more toxic than an equivalent mass dose of fine particles comprised of the same material [10, 11, 25, 26] The increased toxicity of nanoparticles can be attributed to several factors that depend on the nanoparticle under consideration, but for low toxicity, low solubility particles such as those used in the present study. the larger surface area of the particles and resulting oxidative stress appears to be the dominant property [1, 27]. In this study, the effects of particle treatments on type II cells and their potential to induce macrophage migration is investigated.
When treating L-2 cells with the particles used in this study, the concentration of LDH detected in the conditioned medium increased in a dose dependant manner. Maximal LDH release was detected at doses of 1–2 mg/ml of all particles tested. These high doses would never be attained in the lungs under plausible exposure conditions but were used in the study for completeness. The nanoparticles demonstrated a non-significant trend of greater toxicity towards the cells compared to the fine particles. This was indicated by significant increases in LDH concentrations following treatment with both types of nanoparticles. While these results may provide an accurate representation of particle cytotoxicity by measuring LDH concentrations, it is possible that LDH may have been adsorbed on to the surface of the particles following release from the cells. Desai and Richards  and Jones et al.,  both noted how different types of biological molecules could be adsorbed on to the surface of inorganic and mineral dusts. Brown et al.,  also showed extensive binding of Bovine Serum Albumin (BSA) to the surface of nanoparticle carbon black. This may occur when measuring LDH concentrations, especially when taking into account the large surface area of the nanoparticles and the potential for protein adsorption.
Zymosan activated serum has been used as a positive stimulatory agent to induce macrophage chemotaxis in numerous studies with particles [31–33]. Zymosan A is a polysaccharide extracted from yeast cell walls when added to serum, activates the complement system generating large quantities of the macrophage chemoattractant C5a In this study, the J774 macrophages migrated towards ZAS in a dose-dependant manner with significant increases in macrophage migration being observed at ZAS concentrations of 10% and 20% (p < 0.001). Therefore, it was decided, based upon a review of the relevant literature and these results, that 10% ZAS would be an adequate concentration to use as a positive control in future studies using the chemotaxis chamber.
Other macrophage chemotaxis studies have routinely allowed at least 3 hours incubation time to allow macrophages to complete shape change and migration [31–34]. While 3 hours incubation appeared to allow for a significant level of macrophage migration to take place, our results indicated that 6 hours would provide more opportunity for the macrophages to migrate through the filter. This decision took into consideration the fact that future migration studies would be testing a very dilute conditioned medium and as such, macrophages responses could possibly be slower than those observed with Zymosan activated serum.
Prior to conducting particle treatments on L-2 cells, a preliminary experiment was conducted to determine if stimulation by a pro-inflammatory cytokine such as TNFα could induce the type II cells to release macrophage chemoattractants. A significant increase in macrophage migration was observed when the L-2 cells were treated with TNFα at a concentration of 1 ng/ml for 6 hours. A similar macrophage response was not observed in the cell-free supernatants i.e. TNFα at equivalent concentrations that had been incubated for 6 hours in cell-free wells. Although increased migration was observed at TNFα concentrations of 0.1 and 10 ng/ml this was not statistically significant. The 10 ng/ml TNFα concentration may be expected to induce a dose-dependant increase in migration but it is possible that the TNFα concentration could be approaching toxic levels and that this may have actually inhibited migration. The results from this experiment indicate that the L-2 cells were capable of releasing macrophage chemoattractants following pro-inflammatory stimulation. The fact that similar increases in macrophage migration were not observed with the TNFα alone indicated that residual TNFα itself in the medium could not explain the chemotactic response and that chemoattractants released by the L-2 cells into the conditioned medium were responsible. These results correlate well with those published by Standiford et al., , Paine III et al.,  and O'Brien et al.,  who reported that TNFα is able to induce the secretion of chemoattractants from type II epithelial cells. Studies have also shown that IL-9 also induces the release of chemoattractants from bronchial epithelial cells . However, we chose TNFα as the stimulant since it has been shown to be present in the BAL fluid of particle treated animals in many in vivo studies [21, 37, 38] as well as being released by cells in vitro after particle treatment [19, 39].
The results obtained from the LDH experiments indicated that 125 ug/ml of particles would act as a suitable sub-toxic dose for further investigation. L-2 cells were treated at that dose with four different types of particles for 24 hours and the conditioned medium that was generated was tested for potential to induce macrophage migration. The nanoparticle carbon black treatment of the L-2 cells generated a conditioned medium that induced a significant increase in macrophage migration compared to the negative control, whereas none of the other particle treatments had a significant effect on macrophage migration. This would indicate that the carbon black nanoparticles, as well as causing increased cytotoxicity at high dose, are able to stimulate the release of macrophage chemoattractants when exposed to L-2 cells at sub-toxic doses. Cell-free treatments were also conducted to determine whether any soluble substances present on the surface of the particles had the potential to directly induce macrophage migration but no such migration was stimulated. This took into consideration studies by Milanowski et al.,  which showed macrophage chemotaxis towards bacterial and fungal products extracted from organic dust samples. In fact, the migration towards media treated with particles was observed to be slightly lower (although not significantly lower) than the control. This may suggest that the particles may bind or inactivate factors in the media that stimulate cell migration or that they release factors that inhibit migration.
Two nanoparticles were used in these studies, TiO2 and carbon black, but only the carbon black showed any effect in stimulating the release of chemotaxins. This is in accord with previous findings that fine TiO2 was least inflammogenic of a panel of four nanoparticle types, including nanoparticle carbon black, and had least surface free radical activity as measured in a plasmid DNA scission assay . Although both are classified as low toxicity low solubility particles, nanoparticle carbon black has a much greater surface area than nanoparticle TiO2 and this may be a major factor in generation of free radicals in vivo .
Following the experiments using four different particle types, it was decided that further experiments should focus upon the effects of the nanoparticle carbon black on the L-2 cells and the elucidation of the products that were released from the type II cells as a result of particle exposure. SDS-PAGE gels of conditioned medium obtained from L-2 cells treated with 125 μg of either fine or nanoparticle carbon black were run in non-reducing and reducing conditions. In non-reducing conditions, no bands were visible below the 45 kDa molecular weight. However, with the addition of the reducing agent DTT, a higher number of protein bands were noted in the nanoparticle carbon black conditioned medium compared to the medium obtained from the fine carbon black and untreated cells. This indicates that an increased number and concentration of proteins were released from the type II cells into the conditioned medium as a result of the nanoparticle carbon black exposure. One or more of these proteins may account for the increased macrophage chemotactic activity in conditioned medium obtained from such cells. The increased bands were present in the molecular weight range of approximately 14 – 35 kDa. A subsequent experiment where the conditioned medium was centrifuged through molecular weight filters and then tested for chemotactic activity indicated that the substance(s) that induced macrophage migration was located in the 5 – 30 kDa weight range. This is based on the observation that the 5–10 kDa and 10–30 kDa fractions of the conditioned medium induced a significant increase in macrophage migration compared to the negative control. A number of chemotactic proteins such as MCP-1, MIP-1, MIP-2 and rantes are known to be located within these size ranges .
There are important points to take note of when drawing comparisons between in vitro studies and the dosimetry of environmental particle exposure in individuals. Obviously, exposure to a given particulate can vary depending upon a person's occupation, location, method of travel, behaviour etc. Using a miners group as a model, however, Kuempel et al.,  estimated that a coal miner will inhale >350 g of coal dust over a lifetime, and that around 40 g of this will be deposited in the alveolar region of the lung. This figure is not applicable to most people, but draws attention to the potential particle exposure which can occur in a given occupation or workplace. This is a chronic exposure versus the acute exposure used in our study but is important nonetheless. Impaired clearance and protracted exposure of epithelial cells to particles are key factors when attempting to identify the reasons behind the increased toxicity induced by nanoparticles. Although some of the doses used in this study may not be physiologically relevant, they can provide some idea of how type II cells may react in response to acute and, depending on clearance efficacy, sub-chronic exposures to particles.
The phenomenon of rat lung overload in response to high exposures to low toxicity low solubility particles includes, amongst other important pathological sequelae, failure of clearance with subsequent retention of particles . Under conditions of overload there is greatly enhanced contact between particles and epithelium, due to the high rate of particle deposition, exceeding the macrophage's capacity to phagocytose them . Overload is driven by surface area  and so the findings described here provide a mechanism that may help to explain the retention of particle-loaded macrophages in the lung periphery during overload. By this mechanism, the extensive interaction between particle surfaces and epithelial cells characteristic of overload would cause release of chemotaxins that would 'attract' the particle-loaded macrophages to the alveolar region. This gradient would directly compete with the normal chemotactic gradient that would draw such macrophages up the respiratory tract for muco-ciliary clearance. Renwick et al.,  demonstrated that the same types of nanoparticles used in this study, induced an increase in the potential for macrophages to migrate towards a positive chemoattractant, ZAS. In this manscript we have shown that these particles also possess the ability to induce type II epithelial cells to release elevated levels of macrophage chemoattractants. The chemoattractants can stimulate two events, the first being the migration of macrophages towards epithelial cells exposed to particles, as in the case of epithelial cell exposure to nanoparticle carbon black. This event will recruit cells and promote inflammation, but may potentially decrease subsequent clearance if the signal does not subside. In the second instance, a chemotactic gradient may stimulate removal of phagocytic cells out of the lung via the mucociliary escalator, promoting particle clearance. The data shown here demonstrates that nanoparticle carbon black was the most potent tested at eliciting chemotaxin release. This may suggest that such nanoparticles could reverse the normal chemotactic gradient and prevent clearance leading to overload at a lower mass burden than larger particles and this is in fact, amply supported by data (reviewed in ). These are conflicting interpretations of particle effects and require further work to determine the exact pathways governing particle clearance.