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Amorphous silica nanoparticles (SiNPs, 30 and 50 nm) and rhodamine-coated SiNPs (50 nm) were examined for their ability to induce pro-inflammatory responses and cytotoxicity in BEAS-2B cells under different experimental conditions. The SiNPs formed micrometre-sized agglomerates in the absence of bovine serum albumin (BSA) in the culture medium, whereas with BSA (0.1%) they were much less agglomerated. All the SiNPs induced IL-6 and IL-8 responses, as measured by ELISA and real-time PCR. The responses were more marked without BSA and higher for the rhodamine SiNPs than the plain ones. Rhodamine SiNPs were not taken up by cells during a 3-h exposure, even though cytokine mRNAs were up-regulated. In conclusion, agglomerated SiNPs induced more potent cytokine responses than the non-agglomerated ones; either due to the agglomeration state per se or more conceivably to a change in surface reactivity against cellular targets due to BSA. Furthermore, cytokine expression was up-regulated independently of SiNP uptake.

A primary goal of current research on particle-induced health effects is to reveal the critical characteristics that determine their biological effects. Experimental studies have shown that smaller particles induce stronger biological effects than larger particles of similar composition, due to their larger surface area to mass ratio. However, correlation for variations in surface area could not account for variation in biological reactivity among particles of differential composition. Hence, the importance of size and surface area does not override the importance of particle composition. Moreover, different particle characteristics appear to be involved in different biological effects in vitro. Our studies show that mineral particle-induced apoptosis mostly seems to depend on particle size, whereas composition and surface reactivity appeared to be most important for the proinflammatory potential of the particles. The ability of the particles to generate reactive oxygen species in vitro was not correlated with either inflammatory markers or apoptosis, suggesting that other mechanisms are at play. A single, specific component of the mineral particles, explaining the differences in response, has not been identified. In European-wide studies such as the Respiratory Allergy and Inflammation due to Air Pollution (RAIAP) study, particles have been sampled in different locations to study season- and site-dependent variations in responses particles, such as markers of inflammatory and allergic reactions in cells and animals. The data indicate that coarse particles can induce at least as strong inflammatory responses as fine particles. The allergic responses tended to be more associated with the organic fraction (PAH) of particles, whereas the inflammatory reactions seemed to be more associated with metals and endotoxin. Overall, coarse PM was found to have an inflammatory potential similar to fine PM on an equal mass basis. Even though one has to take into account different concentrations in ambient air as well as differences in respiratory system deposition of the size fractions, the potential of coarse particles to induce pulmonary effects should not be neglected.

Type 2 pneumocytes are progenitor cells of alveolor epithelium and important for reepithelialization following lung injury. This study examined the role of persistent versus transient mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase; ERK) in type 2 cell proliferation. Three different types of agents; epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate (TPA), and fetal bovine serum (FBS) induced different patterns of ERK activation. FBS induced a strong and persistent MAP kinase response, whereas the effect of EGF was transient with a strong activation at 5 minutes and only a slight stimulation at 4 hours. The TPA response was more prolonged than the EGF response, but not by far as strong and persistent as the FBS response. Activation by EGF and TPA and the early response induced by FBS were strongly reduced by the MEK inhibitor PD98059. The sustained FBS-induced ERK activation was inhibited by approximately 50%. The total number of cell, the percentage of cells in S and G2/M phase of the cell cycle and the incorporation of 3H-thymidine into DNA were strongly increased in response to FBS, whereas EGF and TPA were without effect. The proliferation was reduced by approximately 50% after pretreatment with PD98059. The results indicate that a persistent ERK activation of a critical size leads to type 2 cell proliferation, and that the proliferative response may also depend on a MEK-independent ERK activation.

CCAAT/enhancer-binding proteins (C/EBP) constitute a family of transcription factors that are involved in regulation of proliferation and differentiation in several cell types. In epithelial lung cells the C/EBP alpha isoform seems to play a role in the regulation of surfactant proteins (SP) and Clara cell specific protein (CCSP), whereas the roles of C/EBP beta and C/EBP delta are unclear. We have examined the protein levels of C/EBP delta in bronchiolar Clara cells and alveolar type 2 cells, and its relation to the expression of lung specific proteins and cell proliferation. The protein expression of C/EBP delta was high in freshly isolated Clara cells compared to type 2 cells. In both cell types C/EBP delta levels increased during culture. Alterations of the levels of C/EBP delta did not correspond with the proliferation levels of Clara cells, but seemed to correspond in type 2 cells. Clara cell secretory protein (CCSP) was highly expressed in freshly isolated Clara cells, in contrast to type 2 cells. SP-D and CYP2B1 were expressed at somewhat higher levels in Clara cells than in type 2 cells, whereas SP-A exhibited highest expression in type 2 cells. During culture the levels of all these lung proteins were strongly reduced. However, compared to with serum we found an increase in CCSP in Clara cell cultures without serum, and this correlated with an increase in C/EBP delta. Overall our in vitro data suggest that C/EBP delta alone is not related to the maintenance of proteins involved in differentiation.

Differential localization of ras proteins and variations in their levels may be of importance during lung growth and differentiation. Abundant cell proliferation occurs during development of the fetal rat lung. As assessed by flow cytometry the proliferative activity declined near birth, followed by a gradual increase in cellular proliferation during the subsequent 8 days and a decline to basal levels by 15 to 18 days of age. During this period of substantial variations in proliferative activity, differences in both the protein content and localization of the different ras proteins were observed. The content of N- and K-ras proteins in lung homogenates increased 5 to 6-fold in rats 20 days or older, compared to fetal levels. The protein levels of the ras proteins remained elevated when cellular proliferation decreased to basal levels. As determined by immunohistochemistry, the localization of N-ras protein was restricted to keratin expressing cells of bronchiolar structures, apparently mainly ciliated cells. In contrast, K-ras was found in alveolar cells, probably type I and type 2 cells. H-ras, but not K- or N-ras, was localized to nonepithelial cells. Thus, different ras proteins were localized to different regions of the lung and increased in abundance during postnatal development.

Bronchiolar Clara cells and alveolar type 2 cells of the lung are known to express relatively high levels of P450 enzymes compared to other pulmonary cells. Populations of enriched type 2 cells and Clara cells were isolated from rat lung by a procedure including lung perfusion, protease digestion, centrifugal elutriation, and differential attachment. Alveolar macrophages were removed by lavage. The purity of the type 2 cell-enriched population was approximately 90%, and the purity of the Clara cell-enriched population was 40-50%. Both type 2 cells and the cells of the Clara cell-enriched population proliferated in culture. CYP2B1 mRNA was expressed approximately to the same level in type 2 cells and the Clara cell-enriched population. The mRNA levels remained roughly constant for both cell types throughout the culture period, except for an early transient reduction. The apoenzyme level of CYP2B1 was 2-3 times higher in freshly isolated cells of the Clara cell-enriched population than in the type 2 cells. Both epithelial cell types showed decreased level of CYP2B1 apoenzyme in culture. The differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated cells and cultured cells suggest the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells. The characterization of specific functions of lung cells in culture, such as P450 gene expression, provides necessary information for the use of the cells in in vitro pulmonary toxicology.