Asthma, rhino-conjunctivitis and allergic sensitization against laboratory animals (LAs) are frequently observed in biomedical research and industry.1,2 Working with LAs is associated with exposure to allergens,3,4 but it also involves exposure to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS)2,5 that further may enhance respiratory symptoms.
In a number of studies, it has been shown that exposure to endotoxin early in life depresses allergic sensitization and development of asthma later in life.6,7 This so called “hygiene hypothesis”8,9 has, however, been widely debated and there are contradictory results.10 In this context, it is interesting that exposure to LPS and other PAMPs often worsen symptoms in individuals who have already developed atopy.11,12
Various PAMPs bind to toll-like receptors (TLRs), a group of pattern recognition receptor belonging to the interleukin (IL)-1R/TLR superfamily.13,14 Ten TLRs have so far been recognized to have essential roles in human13 and activation of TLRs leads to expression of pro-inflammatory cytokines and type I interferons (IFNs) through activation of NF-κB and interferon regulatory factors (IRFs).15–17 Nevertheless, depending on dose, timing of the stimulus and responding cell type, activation of TLRs may also lead to increased production and release of Th2 cytokines such as IL-4, IL-5, and IL-13,18 which are known to have a central role in pathogenesis of atopic disorders and asthma. The IL-1 cytokine family member IL-33 has recently been shown to be the specific ligand for suppression of tumorigenicity 2 (ST2, also known as IL-1R4 and T1), another member of the IL-1R/TLR superfamily. The membrane bound ST2 (ST2L) receptor has, like TLRs, an intracellular toll-IL 1 receptor domain.19,20
Signaling through ST2L by IL-33, lead to a Th2 cytokine response by activation of NF-κB and MAP kinase, which promotes allergic inflammation.21 Furthermore, ST2L has been reported to be a negative regulator of TLR signaling.22–24 Alternate splicing of ST2L generates a soluble isoform of ST2 (sST2) that lacks the transmembrane region and the toll-IL 1 receptor domain.19 Soluble ST2 is known to block the IL-33 signaling in allergic airway inflammation in mice.25
In this study, we investigated whether expression of CD14 and TLRs on blood monocytes and neutrophils, activation markers on B- and T-cells, cytokine profile of blood Th cells and serum levels of sST2 and sCD14 are altered in individuals working in laboratory facilities, a work environment that is associated with high exposure to both allergens and PAMPs. Our aim was to compare two groups who experienced symptoms at work, one sensitized group with positive skin-prick test (SPT) and one non-sensitized group with negative SPT to mouse or rat with two non-exposed groups, one atopic group sensitized to birch pollen and one non-allergic control group.
MATERIALS AND METHODS
Subjects and Study Design
Twenty-seven individuals experiencing symptoms when working with LAs (mice and rats) in a laboratory facility, 12 with allergic symptoms during birch pollen season and 11 healthy non-allergic controls not working with LAs were included in the study (Table 1). All subjects who took anti-histamines were asked to take the last dose not later than 4 days before the visit. The subjects with birch pollen allergy were investigated during birch pollen season. The study was approved by the Karolinska Institute Ethics Committee (Stockholm, Sweden), and all individuals gave their informed consent to participate.
For SPT, a panel of 17 common allergens were used. The SPT were performed on both forearms, and histamine chloride (10 mg/mm, ALK and Allergopharma) was used as a positive control, and the diluent of the allergens (ALK and Allergopharma) as negative control.
Cell Distribution and CD23 and CD25 Staining
Blood collected in ethylene diamine-tetra-acetic acid (EDTA) vacutainer tubes (BD Bioscience, San Jose, California) were stained with a four-color antibody mixture (CD3FITC/CD8PE/CD45PerCp/CD4APC from BD Bioscience) in a TruCOUNT tube (BD Bioscience). Samples were lysed and analyzed on a FACSCalibur™ (BD Bioscience) by using MultiSET™. The results are presented as number of cells/μl blood. To verify proportion of B-cells expressing CD23, blood was stained with titrated amount of anti-CD23 PE, anti-CD45 PerCp, and anti-CD19 APC. To verify the proportion of T-helper cells expressing CD25, titrated amount of anti-CD25 PE, anti-CD3 FITC, anti-CD4 APC, and anti-CD45 PerCp were used (BD Biosciences, San Jose, CA). Samples were analyzed using FACSCalibur™ (BD Bioscience) flow cytometry and CELLQuest™. Gates were set by using fluorescence minus one-stained samples.
TLR and CD14 Staining on Monocytes and Neutrophils
Whole blood (EDTA) was stained with titrated amounts of either anti-TLR2 PE (TLR2.1, eBioscience, San Diego, California), anti-TLR4 PE (HTA125, eBioscience), or anti-CD14 FITC (61D3, eBioscience) together with anti-CD45 PerCp (BD Bioscience). As negative controls, isotype matched antibodies were used. Samples were analyzed using FACSCalibur™ (BD Bioscience) flow cytometry and CELLQuest™. The results are presented as relative median fluorescence intensity (rMFI = monoclonal antibody/matched isotype control).
Intracellular Cytokine Staining
Heparinized peripheral blood in RPMI 1640 containing 2 mM glutamine (1:1 dilution) was stimulated with phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) and ionomycin (1 μM, Sigma–Aldrich) in presence of GolgiPlug (Brefeldin A, BD Bioscience) for 4 hours at 37°C. Stimulated blood was stained with anti-CD4 APC (BD Bioscience). The samples were then lysed and incubated with Cytofix/Cytoperm™ (BD Bioscience Pharmingen) (20 minutes at room temperature) followed by a 10 minutes incubation with Perm/Wash™ (BD Bioscience Pharmingen). The permeabilized cells were stained with anti-IL-4 PE, anti-IFN-γ FITC, anti-IL-2 FITC (BD Bioscience), or anti-IL-13 PE (BD Bioscience Pharmingen). Cells were washed with Perm/Wash and resuspended in CellFIX™ (BD Bioscience). Unstimulated blood was stained using the same procedure as negative controls. Analysis was performed using a FACSCalibur™ (BD Bioscience) and CELLQuest™. The results were present as proportion of cytokine-producing T-helper cells.
Soluble CD14 (sCD14) and sST2 in Serum
For serum analyses peripheral blood was drawn in vacutainer tubes without any anticoagulantia (BD Bioscience). Samples was allowed to clot for 45 minutes and then centrifuged at 3000 rpm 10 minutes × 2. Serum was aliquoted and stored in −70°C until analyzed. Soluble CD14 and sST2 were measured in serum using DuoSet ELISA kits (R&D systems, Europe, Abingdon, UK). All samples had been thawed and frozen once before sST2 was measured. The detection range for sST2 and sCD14 was 15.6 to 2000 pg/mL and 60 to 4000 pg/mL, respectively. For duplicate samples, an intra-assay coefficient <10% was accepted.
Data are given as median with 25th to 75th percentiles unless otherwise stated. Statistical analyses were performed by using Kruskal-Wallis followed by Mann-Whitney U test. A P-value of <0.05 was considered significant.
Among those who experienced symptoms during LA work 19 had positive and 8 had negative SPT to rat or mouse. Thirteen of those with positive SPT to rat also had positive SPT to mouse. In the group with symptoms during birch season all (n = 12) had positive SPT to birch. The control group (n = 11) were negative in SPT for all allergens tested (Table 1).
Cell Distribution, CD23, and CD25 Expression
There were no differences in total amount of cells (five part differential or T-cell subset) and CD25 expression on T-cells between the four groups (Tables 2 and 3). Birch atopics had increased expression of CD23 compared with controls (P = 0.017) and compared with animal workers with positive SPT to rat or mouse (P = 0.016) but not compared with animal workers with negative SPT to rat or mouse (P = 0.35) (Table 3).
TLR and CD14 Expression
The symptomatic group with negative SPT to rat or mouse had increased CD14 expression on monocytes compared with the birch pollen atopic group (P = 0.001), controls (P = 0.001), and the symptomatic group with atopy to LAs (P = 0.006). The group atopic to rat or mouse had increased expression of CD14 compared with birch atopics (P = 0.026) (Fig. 1A). TLR4 expression on monocytes was decreased in birch atopics compared with controls (P = 0.014), those with positive SPT (P = 0.014), and negative SPT (P = 0.013) to rat or mouse (Fig. 1B). There were no differences between the groups regarding TLR2 expression on monocytes and TLR2, TLR4, and CD14 expression on neutrophils (data not shown).
Intracellular Cytokine Expression
There were no differences regarding intracellular IL-4, IFN-γ, IL-2, and IL-13 between the groups (Table 4).
Soluble CD14 (sCD14) and sST2 in Serum
There were no differences regarding soluble CD14 in serum between the groups (Fig. 1C). However, sST2 was increased in the group allergic to birch compared with controls (P = 0.041) and compared with the group with positive SPT to rat or mouse (P = 0.043) but not compared with the group with negative SPT to LAs (P = 0.398). The group with negative SPT to LAs also showed increased levels of sST2 compared with controls (P = 0.006, Fig. 1D).
In this study, it was demonstrated that subjects who experience symptoms, especially those with negative SPT to LAs while working in LA facilities had higher expression of CD14 on peripheral blood monocytes compared with symptomatic, atopic subjects sensitized to birch pollen investigated during birch pollen season and healthy, non-LA-exposed control subjects. It was also shown that blood levels of soluble ST2 were elevated during pollen season in subjects with birch allergy and in workers with LA induced symptoms and negative SPT to mouse or rat compared with a non-atopic non-exposed control group.
As CD14 is critical for the response to LPS26,27 and no difference was revealed between controls and birch pollen allergic subjects during pollen season, it seems most likely that the enhanced CD14 expression is caused by exposure to microbial compound in the animal houses rather than to allergen exposure. We also found that the symptomatic group without atopy to LAs had higher CD14 monocyte expression than those with positive SPT to LAs. It could be assumed that this finding reflects a protective role in development of allergy to LAs as a specific polymorphism in the promoter region of CD14 (C-159T) has been shown to prevent atopy and is associated with increased in vitro expression of CD1428 and increased serum levels of sCD14.29 It is, however, not clear whether this gene variation occurs more often in the symptomatic group without atopy to rat or mice than in sensitized subjects. Nevertheless, except the increased surface expression of CD14 on monocytes we did not observe increased levels of sCD14 in this group, indicating that this is probably not the case.
The ST2 receptor is expressed by several cell types on activation but is constitutively expressed by Th2 effector cells30 and mast cells.31 These cell types are involved in allergic inflammation and asthma and there are indications that ST2 is of importance in these conditions.32,33 In line with our results, it has previously been shown that sST2 is elevated in serum from atopic patients during asthma exacerbation and that sST2 levels correlate with exacerbation severity.34 Recently, Ali et al reported higher serum levels of sST2 in children during acute asthma exacerbation and a relationship between sST2 levels and blood eosinophil count. They also showed that specific polymorphism in the ST2 gene was associated with differences in asthma phenotypes and asthma severity.32 Thus, the increase of serum sST2 in the birch atopic group during pollen season in this study most likely reflects an ongoing allergic airway inflammation due to birch pollen exposure.
It was also demonstrated that sST2 blood levels were elevated in animal workers who were SPT negative to rat or mice (but not those who were SPT positive) compared with controls. A possible explanation to this finding may be differences in exposure, a hypothesis that is supported by the finding of differences in CD14 monocyte expression between LA SPT positive and negative individuals. Allergic subjects working in animal facilities are acquainted with their allergy to LAs and they often take measures to minimize exposure. Such measures are use of protection masks, gloves, clothes, working on down flow benches, and use of airflow masks. These measures minimize the exposure both to allergens and PAMPs including LPS.
It is not clear whether the increased levels of sST2 in the SPT negative group of symptomatic animal workers depends on activation of the IL-33/ST2 system caused by allergens, non-allergic pro-inflammatory stimuli or both. Tajima et al showed that a combination of pro-inflammatory stimuli and Th2 cytokines have a synergistic effect on ST2 transcription and production.35 The elevated levels of sST2 observed in the birch atopic group most likely comprise a negative regulation to the acute exposure to high levels of birch pollen during birch pollen season. This hypothesis is supported by the finding that sST2 has the capacity to block IL-33 signaling and thereby interrupt allergic sensitization and inflammation25 and that interruption of the ST2/IL-33 pathway is important in resolution of the allergic inflammation.33 This may indicate that the elevated ST2 levels, observed in the group of workers without rat or mouse allergy, may have a protective role in sensitization to LAs. On the other hand, it has been shown that both membrane bound ST2 and sST2 have the ability to inhibit release of Th1 cytokine in mice models and by monocytes/macrophages,22–24 which may favor a Th2 response possibly enhancing the risk for atopic sensitization. The increased levels of sST2 in animal workers negative in SPT might then be an early inflammatory marker for sensitization.
In conclusion, soluble ST2 were increased in serum of birch atopics with an ongoing allergic inflammation, and soluble ST2 and CD14 expressions on blood monocytes were elevated in individuals who experienced symptoms from the respiratory tract when working in laboratory facilities but had negative SPT to rats or mice. These findings indicate that CD14 seems to respond to non-allergic stimuli whereas sST2 respond to acute allergen exposure in atopic individuals as well as to PAMPs.
We thank Åsa Andersson, Kristin Blidberg, Anne Renström, and Britt-Marie Sundblad for excellent assistance.
This work was supported by Swedish Heart-Lung Foundation, Swedish Asthma and Allergy Association, and Swedish Council for Working Life and Social Research.
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