Assessment of the Total Inflammatory Potential of Bioaerosols by Using a Granulocyte Assay

Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by traditional assays for dust, endotoxin, fungal spores, (1→3)-β-d-glucan, total number of bacteria, the enzyme N-acetyl-β-d-glucosaminidase (NAGase; primarily originating from fungi), Aspergillus fumigatus, and mesophilic and thermophilic actinomycetes; the samples were also assayed for TIP. In a multilinear regression four factors were significant for the TIP values obtained: endotoxin (P < 0.0001), fungal spores (P < 0.0001), (1→3)-β-d-glucan (P = 0.0005), and mesophilic actinomycetes (P = 0.0063). Using this model to estimate TIP values on the basis of microbial composition, the correlation to the measured values was r = 0.91. When TIP values obtained in the granulocyte assay were related to the primary working area, we found that bioaerosol samples from personnel working in straw storage facilities showed high TIP values (≈50 times the TIP of unstimulated controls). In contrast, bioaerosol samples from personnel with work functions in offices or laboratories showed low TIP values (≈5 times the TIP of the unstimulated control). This indicates, as expected, that these areas were less contaminated. In conclusion, the granulocyte assay reacts to multiple contaminants in the environmental samples and can be used to obtain a measurement of TIP. Therefore, potential occupational health effects related to inflammation of the airways in a working environment can be estimated using this assay.For several years reports have related increased prevalence of respiratory symptoms to the exposure to bioaerosols containing, e.g., organic dust particles, actinomycetes, endotoxin, and fungal spores. Occupational health effects of bioaerosols have been reported in swine confinement houses and poultry farms as well as during hay handling. Airway diseases are frequent occupational disorders among farmers in many countries around the world (19, 23). During mechanical handling, biofuels such as straw and wood chips release high amounts of various microbial components such as, e.g., actinomycetes, fungal spores, and endotoxin (11, 25). Studies of personnel exposure to bioaerosols at biofuel plants reveal high concentrations of different microbial components and exposure levels higher than the suggested occupational exposure limits (10).Exposure limits or suggested exposure limits are usually based on studies where traditional microbial quantification methods have been applied and are normally related to the concentration of a single contaminant (e.g., endotoxin) (17) or to gravimetric measures of dust. Furthermore, it is recognized that exposure to various microbial components may cause inflammation in the airways (4, 5, 15, 16, 20, 34, 35). Consequently, both the quantitative and the qualitative composition of bioaerosols may be of importance. In optimal settings, risk assessment of bioaerosols is based on the presence and concentration of a variety of microbial components. However, such analyses are tedious and costly. Therefore, an assay taking multifactorial contaminations into account may be highly relevant, and ideally this assessment should also correlate to airway inflammation. The objective of this work is to find a rapid and cost-efficient alternative to microbial analyses in characterizing bioaerosols from occupational settings.Normally, airways and alveoli contain a relatively small number of granulocytes, but the pulmonary vasculature represents the largest reservoir of granulocytes in the human body. From this pool, granulocytes can be rapidly recruited upon microbial challenge (26). This recruitment is crucial for the immune response of the host against pathogens. Consequently, we speculate that the production of reactive oxygen species (ROS) of granulocytes can be used as readout for bioaerosol exposure. However, isolated granulocytes are short-lived, with a half-life of only a few hours (33). For this reason a granulocyte-like cell line can represent a practical alternative to freshly isolated cells.In 2006 a granulocyte assay based on a cell line was developed mainly to assess microbial contamination in medicines (32). The assay was shown to be very sensitive to a wide range of microorganisms (32) and severalfold more sensitive to fungi than monocyte assays (e.g., the Mono Mac 6 assay) (18, 32). Furthermore, the assay showed sensitivity very similar to that of freshly isolated granulocytes toward a variety of microorganisms and microbial components (our unpublished observations). This indicates that the granulocyte assay is suitable to assess the inflammatory potential of bioaerosols or similar environmental samples.The granulocyte assay is based on differentiated HL-60 cells. To assess the immunomodulatory effect of a given environmental sample, the production of ROS from the granulocyte-like cells is measured in a luminol-dependent chemiluminometric assay. The induced ROS production is used as a measure of the total inflammatory potential (TIP) of the sample. The term TIP as described herein refers to the inflammatory effects induced in the granulocyte assay by the bioaerosols tested. These effects can be induced by both viable and dead microorganisms as well as related cell wall debris. Furthermore, the term covers inflammatory effects induced by other organic and inorganic components of the bioaerosols, such as pollen, particles, viruses, soot, etc., that are known to have or speculated to have inflammatory effects in the granulocyte assay.To examine the potential of the granulocyte assay to measure the TIP of an environmental sample, we examined 129 bioaerosol samples collected at 22 biofuel plants. In occupational settings the presence of fungi, Aspergillus fumigatus, β-glucan, endotoxin, actinomycetes, and dust has been related to airway symptoms (4, 5, 15, 16, 20, 34, 35). Therefore, we have quantified these components in the sampled bioaerosols. Furthermore, we have quantified N-acetyl-β-d-glucosaminidase (NAGase) activity because this enzyme is associated with fungi and has been shown to correlate significantly with the total number of fungal spores counted by microscopy (9, 14). Our hypothesis is that the TIP values obtained in the granulocyte assay can be used as a rapid way to evaluate the bioaerosol exposure and thus could indicate where a more extensive characterization of microbial parameters would be necessary.