Qualitative Immunodetection of Hsp70 in Nasal Samples of Children With Allergic Rhinitis

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Allergic rhinitis (AR) is the most common chronic disorder of the upper airway in the pediatric population and is thought to be a relevant risk factor for the development of asthma. Many studies have demonstrated that Heat shock protein 70 (Hsp70) plays an important role in the pathogenesis of several allergic diseases such as AR and asthma, by modulating immunological processes and inflammatory responses.^1,2 Recently, by using quantitative qPCR analysis we demonstrated that stress-inducible Hsp70 transcripts were differentially expressed in the nasal mucosa cells of pediatric patients with different phases of AR clinical activity, showing upregulation of Hsp70 mRNA in symptomatic patients compared to asymptomatic patients. These findings and the correlation between Hsp70 in nasal mucosa and FeNO in lower airways suggested a potential role for inducible Hsp70 as a predictive nasal biomarker that can define the risk of asthma development in children with AR. ³

In this study, the qualitative analysis of inducible Hsp70 on nasal samples of pediatric patients with AR is carried out to determine which cell source expresses the protein, providing new insights into AR pathogenesis in children. Only a single study has employed nasal cytology in adult patients with seasonal allergic rhinitis to examine the presence of Hsp70 in the nasal mucosa cells. ⁴

Twelve children (age range 8–12 years) with a diagnosis of AR, who were in follow-up as outpatients, were enrolled following the criteria described in Fagotti et al. ³ The collection of nasal samples was performed using a non-invasive method. ³ Harvested cells were processed using the cell block method and the collodion bag technique. Immunohistochemistry was performed using a monoclonal antibody against the stress-inducible form of Hsp70 (ab 47455 Abcam, Cambridge, UK) at 1:200 dilution.

Qualitative immunocytological analysis showed a co-mparable localization pattern of inducible Hsp70 in all AR patients, both symptomatic and asymptomatic. Positive nuclear and cytoplasmic Hsp70 staining was observed in resident epithelial cells of the nasal mucosa, particularly in columnar ciliated and striated cells (Figure 1(A)), and in muciparous goblet cells (Figure 1(B)). No Hsp70 expression was observed in inflammatory cells, such as polymorphonuclear cells and lymphocytes, against the background of mucus (Figure 1(C)).

Figure 1.

(A-C) Immunolocalization of inducible Hsp70 in nasal mucosal cells from children with allergic rhinitis. Columnar ciliated cells (A, thin arrow), striated cells (A, thick arrow), and goblet cells (B, C, arrowhead) show a positive immunostaining reaction. No immunoreactivity is detected in inflammatory cells (C, asterisk). Bar: 50 µm.

To our knowledge, this is the first report highlighting in children with AR which cytotypes were positive for stress-inducible Hsp70. Many studies reported both pro-inflammatory and anti-inflammatory effects of Hsp70 in upper and lower airway inflammation. ² It has been hypothesized that during asthma inflammation, intracellular Hsp70 is involved in stress-induced cellular responses, playing an important function in degrading denatured proteins and damaged organelles. In addition, Hsp70 also acts as a “danger” molecule capable of modulating immune and inflammatory responses when it is locally released from stressed cells into the extracellular space. ² In the children we examined, the immunoreactivity of the resident epithelial cells suggests that inducible Hsp70 could have a protective role in epithelial cells affected by potential damage following airway inflammation. However, further investigations will need to determine if epithelial cells, through the release of Hsp70, are also actively involved in modulating the local immune response. This study confirms the utility of nasal cytology as a non-invasive instrument in pediatric patients to investigate nasal diseases, such as allergic rhinitis. ⁵