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Keywords:

  • rhinitis;
  • occupational rhinitis;
  • nasal lavage;
  • nasal challenge;
  • neurotrophins;
  • brain-derived neurotrophic factor

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Background

Neurotrophins may play a role in the pathophysiology of allergic occupational rhinitis (OR). We sought to investigate whether an immediate allergic reaction that induces nasal inflammation is also able to induce changes in levels of brain-derived neurotrophic factor (BDNF) in nasal lavage (NAL) fluid from patients with allergic OR.

Methods

Ten patients sensitized to flour underwent control and active specific inhalation challenge (SIC) on consecutive days. Nasal response to SIC was monitored with acoustic rhinometry and symptoms recording. NAL was performed before and 30 minutes, 6 hours, and 24 hours after control and active challenge for the assessment of levels of BDNF and inflammatory cells in NAL fluid.

Results

In contrast to control day, flour challenge induced immediate clinical reactions in all subjects. After flour challenge, a significant increase in levels of BDNF in NAL fluid was observed at 6 hours after challenge (p < 0.05). Also, a significant increase in the number of eosinophils in NAL fluid at 30 minutes (p < 0.01), 6 hours (p < 0.01), and 24 hours (p = 0.05) postchallenge was observed. Also, levels of BDNF in NAL fluid were significantly higher at 30 minutes after flour challenge (p = 0.02) in comparison to levels on the control day at the same postchallenge time. A marginally significant positive correlation between BDNF levels and eosinophil counts at 30 minutes (r = 0.60, p = 0.06) and at 6 hours (r = 0.50, p = 0.08) after flour challenge was noted.

Conclusion

We showed that BDNF is released in nasal fluid after SIC with flour. Results support the suggestion that neurotrophins may play a role in the pathogenesis of allergic OR.

Occupational rhinitis (OR) is a type of rhinitis caused by exposure to occupational agents through well-established allergic mechanisms and nonallergic mechanisms that have not yet been determined. OR patients exhibit significantly increased reactivity against a wide spectrum of allergens and irritants, resulting in expression of nasal symptoms such as itching, sneezing, and rhinorrhea. Presently, there is rising interest in the potential important role of the nervous system in pathogenic mechanisms and clinical manifestations of respiratory allergic diseases such as allergic rhinitis and asthma.[1] The neurotrophins family, comprising nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4, are key regulators of neuronal activity in humans. The neurotrophins have gained increased attention due to their potential role in allergic rhinitis, participating in nasal hyperresponsiveness and nasal airway inflammation.[2, 3] Particularly, increased levels of the neurotrophin BDNF have been found in the peripheral blood of allergic rhinitis patients. Also, a study involving nasal allergen challenge showed upregulation of BDNF in nasal mucosa biopsies 24 hours after nasal allergen challenge in patients with allergic rhinitis as compared to controls.[4] Also, BDNF appears to have a role in the observed eosinophilic inflammation characteristic of allergic respiratory diseases such as asthma and allergic rhinitis. This effect is exerted by direct interaction with the neurotrophin receptors on the surface of eosinophils.[4] Human peripheral eosinophils are also capable of producing, storing, and releasing neurotrophins.[5] Further, BDNF increases the release of eosinophil protein X of peripheral blood eosinophils of allergic rhinitis patients.[6]

There is limited data on changes in levels of neurotrophins in nasal lavage (NAL) fluid following allergen challenge. A significant increase in NGF concentrations in NAL fluid was observed following allergen challenge.[7] The objective of this study was to assess the effect of specific inhalation challenge (SIC) with flour on levels of BDNF in NAL fluid. In addition, we wanted to assess whether the SIC methodology was a useful tool to examine a potential role of neurotrophins in allergic OR.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Subjects

We recruited 10 subjects sensitized to flour with a known diagnosis of allergic OR and occupational asthma. In all cases the diagnosis of OR was confirmed in a laboratory at the Department of Chest Medicine, Hôpital du Sacré-Coeur de Montréal. Table 1 shows clinical characteristics of patients. All subjects were atopic as determined by positive skin prick tests to common aeroallergens and flour. Most of them were nonsmokers or ex-smokers (8/10) with a mean duration of work exposure to flour of 8.9 years. At the time of the study, subjects had been removed from the workplace and rhinitis and asthma symptoms were stable in all patients. The study protocol was approved by the hospital ethics committee.

Table 1. Clinical characteristics of subjectsa
  1. a

    Values are number of subjects unless otherwise indicated.

  2. ES = ex-smoker; F = female; FEV1 = forced expiratory volume in 1 second; M = male; NS = nonsmoker; PC20 = concentration of methacholine that caused a 20% fall in FEV1; S = smoker.

Subjects10
Sex, M/F10/0
Age, years, mean ± SD39.1 ± 9.1
Atopy, present/absent10/0
Smoking, S/ES/NS4/4/2
Duration of exposure at work, years, median (25th and 75th percentile)8.9 (8.0–20.0)
FEV1 % predicted, mean ± SD96.5 ± 13.7
PC20 ≤ 16 mg/mL7/10

SIC

For this study, all subjects underwent standardized SIC with monitoring of nasal responses on 2 consecutive days following the same protocol. As described,[8] on the first day subjects were exposed to a nonspecific high-molecular weight (HMW) agent (lactose) and the next day they were exposed to flour by recreating working conditions in an isolated challenge chamber according to international recommendations.[9] The total exposure time on both days was 30 minutes.

Total nasal volume and nasal symptoms were measured before and serially for 6 hours after exposure by acoustic rhinometry and a visual analogue scale, respectively. Nasal fluid was collected by NAL prechallenge and at 30 minutes, 6 hours, and 24 hours postchallenge during both challenge days.

Acoustic rhinometry and visual analogue scale

Acoustic rhinometry was performed according to a standardized procedure.[10] An acoustic rhinometer (Hoods Laboratories, Pembroke, MA) was used to measure the nasal volume between 2 and 5 cm into the nose (Vol2–5); Vol2–5 was selected as the endpoint for this study to better reflect mucosal changes during the challenge.[10, 11] According to published studies and recommended guidelines for the interpretation of nasal challenge test,[12, 13] a decrease in Vol2–5 after challenge >25% from baseline values represents a positive response to challenge. The subject's subjective bilateral perception of nasal congestion was quantified using a 100-mm visual analogue scale (VAS).

NAL and quantification of BDNF and inflammatory cells

NAL was performed using a modified 10-F standard silicone Foley catheter (Teleflex Medical GmbH, Ruesch, Germany).[14] The tip of the catheter was cut distal to the balloon to avoid touching the septum and the anterior tip of the inferior turbinate. Then, the catheter was introduced about 1.0 cm into the vestibulum and inflated to form a seal. Ten milliliters (10 mL) of warm normal saline solution (0.9%) was instilled into the nose via a 10-mL needleless syringe connected to the catheter and then aspirated after 30 second. The entire procedure involved 3 instillation/aspiration cycles. The balloon was then deflated and the procedure was repeated on the other nostril. The NAL fluid from both nasal cavities was pooled in the same plastic container and kept on ice until processed.[15]

Two NALs were performed prechallenge; the first was intended to remove preexisting mediators and the second to obtain baseline BDNF and inflammatory cell levels for comparison with postchallenge values. Within 2 hours the sample was measured and centrifuged at 3300 revolutions per minute (rpm) (1705 g) for 8 minutes at 4°C and the supernatant was frozen at −80°C for future analysis. The pellet was resuspended in 0.5 mL phosphate-buffered saline (PBS) containing 0.1% wt/vol bovine serum albumin. Cytocentrifuge preparations were made by using 100 μL of the remaining resuspended cell suspension. The preparation was centrifuged at 450 rpm (32 g) and slides were stained with Wright-Giemsa for differential cell counting. BDNF was measured in NAL fluid using the RayBio Human BDNF ELISA kit (Lucerna-Chem AG, Luzern, Switzerland) according to the manufacturer's instructions. The detection limit of the assay was less than 80 pg/mL.

Statistical analysis

The nonparametric Friedman test was used to test the significance of changes in nasal levels of BDNF and inflammatory cells after control and active challenge. Related-samples Friedman's 2-way analysis of variance by rank test was used to compare changes in nasal volume and NAL fluid volume at different challenge times. All differences within sets of paired data representing before and after challenge measurements were analyzed using the nonparametric Wilcoxon signed rank test. Correlations between levels of BDNF and inflammatory cells at different challenge times were evaluated by means of the Spearman rank correlation coefficient. The analysis was performed using the SPSS 18.0 statistical package (SPSS, Inc., Chicago, IL).

Results

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Clinical response to SIC

In contrast to control challenge, flour challenge induced rhinitis symptoms and objective nasal congestion in all subjects. During the control day the mean ± standard deviation (SD) maximum percentage fall in nasal volume (Vol2–5) as compared to baseline was 15.0% ± 10.4%. After challenge with flour the maximum percentage fall was 38.7% ± 8.6% for the whole group of subjects. In addition, flour challenge induced subjective nasal congestion. On the control day, the median (25th and 75th percentiles) VAS rating at baseline was 0.3 (0.0–2.7) and no increase was observed after control challenge. On the active challenge day, the VAS rating at baseline was 0.3 (0.0–2.3), whereas the median highest VAS rating was 3.4 (0.0–4.9) at 10 minutes after challenge with flour.

Inflammatory cells in NAL fluid

After control challenge there was a significant increase in the number of eosinophils in NAL fluid at 30 minutes postchallenge (p = 0.02) (Fig. 1). After flour challenge, a significant increase in the number of eosinophils during the early phase reaction to challenge (at 30 minutes, p = 0.008), and at 6 hours (p = 0.008) and 24 hours (p = 0.03) postchallenge was observed (Fig. 1). During control and flour challenge, increases in the number of neutrophils and macrophages were not significant (Fig. 1).

image

Figure 1. Changes in inflammatory cells in nasal lavage during control (left) and flour challenge (right) days. *p < 0.05; **p < 0.01. Circles are outliers.

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BDNF in NAL fluid

After control challenge, BDNF levels in NAL fluid did not increase when compared to prechallenge levels (Fig. 2). In contrast, after flour challenge, a significant increase in levels of BDNF in NAL fluid was observed at 6 hours compared to prechallenge levels (p < 0.05) (Fig. 2). There were nonsignificant effects at 30 minutes (p > 0.05) and 24 hours (p > 0.05) postchallenge (Fig. 2). Individual changes in levels of BDNF in NAL fluid from baseline to 6 hours postchallenge are shown in Figure 3.

image

Figure 2. Changes in nasal levels of BDNF in nasal lavage during control (A) and flour challenge (B) days. *p < 0.05. BDNF = brain-derived neurotrophic factor.

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image

Figure 3. Individual changes in nasal levels of BDNF from baseline to 6 hours postchallenge during control (A) and flour challenge (B) days. *p < 0.05. BDNF = brain-derived neurotrophic factor.

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These results showed an increase in BDNF levels in 8 of 10 subjects and a mild decrease in 2 subjects at 6 hours postchallenge. As shown in Figure 3, a clear increase in BDNF levels was observed in 3 of 10 patients. Subjects showing the greater increases in BDNF levels were not those showing the greater changes in VAS scores and nasal volume; however, the observed changes were considered clinically significant. The analysis at 6 hours postchallenge showed moderate nonsignificant correlations between changes in acoustic rhinometry and BDNF levels (r = 0.45, p = 0.2) and changes in VAS levels and BDNF levels (r = 0.38, p = 0.4).

We further compared BDNF levels from the control and flour challenge days measured at each postchallenge time (30 minutes, 6 hours, and 24 hours) to assess whether or not BDNF levels were higher after flour challenge. Data was analyzed by first calculating a ratio that accounted for prechallenge differences in concentrations of BDNF (eg, BDNF level at 30 minutes postchallenge vs prechallenge level; BDNF level at 6 hours postchallenge vs prechallenge level; and BDNF level at 24 hours postchallenge vs prechallenge level) and then comparing the ratios calculated for the control and flour challenge days at each postchallenge time. This analysis showed a statistically significant difference at 30 minutes postchallenge (p = 0.02), indicating higher levels of BDNF after flour challenge compared to control challenge. After flour challenge, a trend toward higher levels of BDNF at 6 hours compared with control day levels was observed but did not reach statistical significance (p = 0.06). Figure 4 shows that at 30 minutes after flour challenge, the ratios from all 10 subjects were higher than during the control day. At 6 hours after flour challenge, the ratio was higher in 8 subjects and lower in 2 subjects compared to the control day.

image

Figure 4. Scatter plots showing BDNF ratios for each subject during the control (x axis) and flour (y axis) challenge days at 30 minutes (A) and 6 hours (B) postchallenge. The reference line connects the lowest and highest ratio observed during the control day. At 30 minutes after flour challenge, the BDNF ratio from all 10 subjects was higher compared to control day values indicating higher levels of BDNF in NAL after flour challenge. At 6 hours after flour challenge, the ratio was higher in 8 subjects and lower in 2 subjects compared to control day values. BDNF = brain-derived neurotrophic factor; NAL = nasal lavage.

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A marginally significant positive correlation between BDNF levels and eosinophils counts at 30 minutes (r = 0.60, p = 0.06) and at 6 hours (r = 0.50, p = 0.08) after flour challenge was noted while a negative significant correlation between BDNF levels and neutrophil counts was observed at 30 minutes after flour challenge (r = −0.64, p = 0.04).

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Neurotrophins such as NGF and BDNF participate in the allergic response by upregulating the release of sensory neuropeptides, including tachykinins such as substance P (SP), neurokinin A (NKA), and neurokinin B (NKB).[16] The biological effects of these neuropeptides play a role in the initiation of neurogenic inflammation inducing edema and vasodilatation, which may manifest clinically as nasal obstruction.[17] Neurotrophins and tachykinins also have immunomodulatory effects on inflammatory cells involved in the pathogenesis of allergic diseases. In addition, immune cells themselves are a source of neurotrophins.[3]

A previous study showed a functional relationship between neurotrophins and features of allergic rhinitis after allergen challenge.[4] This study showed that BDNF can be recovered in significantly increased concentration in nasal fluid after SIC in patients with flour-induced allergic OR. The increased secretion of BDNF was associated with elicitation of rhinitis symptoms and nasal eosinophilic inflammation. In this study, subjects acted as their own control and the observed changes in levels of BDNF in nasal fluid were not observed after challenge with a nonspecific HMW agent. Thus, it is likely that the source of the observed increase in levels of BDNF in nasal fluid after flour challenge was the nasal mucosa during the induced allergic reaction.

The results of this study add to a growing body of research suggesting a role of neurotrophins in allergic rhinitis. A potential role in allergic rhinitis was first suggested for NGF, which is probably the most extensively studied member of the neurotrophin family. It was shown that in the nasal mucosa, the submucosal glands, the mucous cells of the epithelial lining and eosinophils are sources of NGF.[18, 19] Also, increased levels of NGF in NAL fluid[7] and increased expression in nasal mucosa has been demonstrated in allergic rhinitis patients after allergen challenge.[4] By contrast, BDNF neurotrophin protein has been relatively less studied; however, there is now accumulating evidence suggesting a role of this protein in contributing to allergic airway inflammation.[20] Animal models have shown that the production of BDNF by airway epithelial cells is upregulated in allergic airway inflammation.[21, 22]

The involvement of BDNF in nasal airway inflammation was first revealed in a study that showed that the production and secretion of BDNF were markedly increased in response to proinflammatory cytokines in human epithelial cells isolated from nasal polyps and middle turbinates.[23, 24] To our knowledge, only 1 study has examined modulation of BDNF protein after allergen challenge.[4] The study showed increased nasal expression of BDNF 24 hours after nasal allergen challenge in allergic rhinitis patients when compared to controls, suggesting a potential role of BDNF in the late phase of the nasal allergic response. Overall, our findings concur with this study, showing increased levels of BDNF in nasal fluid in the late phase of the nasal allergic response. However, in contrast to that study, the enhanced release of BDNF in our study was observed at 6 hours after challenge whereas BDNF levels at 24 hours after challenge were not increased. Our study differs from that study in many ways, which may account for this observed difference. First, subjects were challenged with an occupational allergen rather than with a common aeroallergen. Second, the SIC methodology differs from the standard nasal allergen challenge test because it resembles a more natural allergen exposure. Third, we assessed levels of BDNF in NAL fluid rather than in nasal mucosa specimens.

Eosinophils play an important role in the pathophysiology of allergic rhinitis. In previous studies,[25, 26] we have also demonstrated their involvement in allergic OR. There is good evidence supporting a role of neurotrophins contributing to eosinophilia in allergic disorders.[27] It has been suggested that neurotrophins may exert immunomodulatory functions on eosinophils by influencing the recruitment and activation of these cells into the nasal mucosa. In the present study, we observed a significant increase in the number of eosinophils in NAL fluid at 30 minutes, 6 hours, and 24 hours after flour challenge as compared to prechallenge values. The maximum increase was observed at 30 minutes after challenge; on the other hand, BDNF levels were found to be significantly increased only at 6 hours postchallenge. Therefore, in the present study, the observed temporal relationship between changes in nasal levels of BDNF and eosinophils after flour challenge does not support a potential role of BDNF influencing recruitment and activation of eosinophils into the nasal mucosa. However, there is also evidence that eosinophils are a source of neurotrophins,[3, 27] which would be more in accordance with our findings.

This study has limitations, including the small size, the lack of complementary laboratory analysis demonstrating an increase in nasal BDNF levels, and the lack of data on other neurotrophins and tachykinins that may play also a role in neurogenic inflammation. This prevents us from speculating on pathophysiologic aspects linking our findings with neurogenic inflammation pathways.

Despite the study limitations, we believe that our results extend the findings of a potential role of BDNF in allergic rhinitis in the general population to BDNF's role in work-related rhinitis; our results also confirm the value of NAL for studying the kinetics of inflammatory mediators in patients with OR.

Conclusion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References

In summary, our findings support the hypothesis that neurotrophins may contribute to nasal allergic inflammation in patients with allergic OR. Also, the SIC methodology appears to be a useful tool to assess pathogenic mechanisms of work-related rhinitis.

References

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. References