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\begin{document}

\title{Allergic inflammation after allergen challenge -- insights from the
tissue}



\author[1]{Frauke Prenzler}%
\author[2]{Thomas Tschernig}%
\author[3]{Katherina  Sewald }%
\author[4]{Tibor Veres}%
\author[5]{Susanne Rittinghausen}%
\author[6]{Norbert Krug}%
\author[7]{Jens Hohlfeld}%
\author[3]{Armin  Braun }%
\affil[1]{Fraunhofer-IEM}%
\affil[2]{Saarland University}%
\affil[3]{Fraunhofer Institute for Toxicology and Experimental Medicine, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre for Lung Research (DZL)}%
\affil[4]{NIH}%
\affil[5]{Fraunhofer-Institut für Toxikologie und Experimentelle Medizin ITEM}%
\affil[6]{Fraunhofer ITEM}%
\affil[7]{Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover - Germany}%


\vspace{-1em}



  
  \date{May 31, 2024}


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\textbf{Allergic inflammation after allergen challenge -- insights from
the tissue}

To the Editor,

Limited data exist on the infiltration of eosinophils in direct response
to allergen exposure in asthmatic patients. The experimental procedure
of segmental allergen provocation (SAP) in mild asthmatic subjects is an
extremely valuable study tool to investigate mechanisms of bronchial
asthma in patients in general and in particular for the role of
eosinophils. In this procedure, BAL and bronchial mucosa can be analysed
simultaneously after the induction of allergic inflammation. Older
studies yielded data on different time points after the challenge with
contradictory results. Eosinophils were studied in mucosa and airway
lumen of mild asthmatics undergoing segmental allergen
provocation\textsuperscript{1,2}. Here, eosinophils and their release
products were investigated in thick sections of the bronchial biopsies.
Subject data, methods and detailed results are given in the supplement.
All subjects showed a clear eosinophilic response in the airway lumen
and mucosa 24 hours after the challenge (Figure 1, S1). The data on
neutrophils were inconclusive (Figure 1, S2). There was an increase of
eosinophils in the mucosa and in the BAL. In the BAL the concentrations
of IL-5 and ECP were elevated (supplement). In the mucosa ECP stained
areas were found elevated as well as signs of eosinophil activation and
degranulation (Figure 2). ECP~staining was associated with cellular
structures, small granules or dispersed over a large area beneath the
epithelium. The degree of released mediators into the tissue is an
important parameter investigating new drugs for asthma. In the presented
study not cell associated, free ECP positive granules were seen at
baseline but only to a small extent. However, after allergen challenge
the ECP volume was significantly increased and only few distinct cells
could be detected being positive for ECP. These results suggest that in
human tissue eosinophils degranulate to a significant amount after a
single allergen challenge and therefore release all their toxic content
into the surrounding tissue. This is in contrast to animal models of
asthma where eosinophilic degranulation after allergen challenge does
not occur extensively \textsuperscript{3,4}. In the present study,
subjects with a strong IL-5 reaction in BAL showed a strong eosinophilic
response in the lumen. However, BAL IL-5 levels did not correlate with
eosinophil numbers in the mucosa or volume of ECP positive surfaces in
the mucosa (Figure S3). It is a long known fact that IL-5 levels in the
BAL correlate highly with absolute numbers of eosinophils in the same
compartment~\textsuperscript{5}. In the present study the allergic
reaction is comparable to those of other studies. The missing
correlation between IL-5 in the BAL and numbers of tissue eosinophils
showed that the relationship between both compartments is not as simple
as assumed. Here, the data for the volume of ECP positive surfaces may
give an important hint. Interestingly, the volume of ECP positive
surfaces was inversely correlated with TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-8. TNF-\selectlanguage{greek}α \selectlanguage{english}enhances
migration of eosinophils from mucosa to lumen shown in an in vitro cell
culture model \textsuperscript{6}. Therefore, one possible explanation
is the more TNF-\selectlanguage{greek}α \selectlanguage{english}is found in the BAL the less activated eosinophils are
found in the mucosa. In conclusion, the findings in subjects with mild
asthma are in alignment with other published results and suggest that 1)
human tissue eosinophils release their granules in non-provoked state,
and 2) toxic content of these cells is significantly released into the
surrounding tissue after a single allergen challenge whereas the
distribution and the degree of activation and degranulation of
eosinophils differs widely between subjects. Many eosinophils in the
airways indicate many eosinophils in the mucosa. Three-dimensional
analysis in thick tissue sections using confocal microscopy is a
valuable tool in the investigation of bronchial biopsies from patients
suffering from bronchial asthma.

(Abbreviations: BAL=bronchoalveolar lavage, ECP=eosinophilic cation
protein, IL=interleukin, MBP=major basic protein, NE=neutrophilic
elastase, SAP=segmental allergen provocation, TNF=tumor necrosis factor)

References

1. Erpenbeck VJ, Hagenberg A, Dulkys Y, et al. Natural porcine
surfactant augments airway inflammation after allergen challenge in
patients with asthma. \emph{Am J Respir Crit Care
Med.}2004;169(5):578-586.

2. Schaumann F, Muller M, Braun A, et al. Endotoxin augments myeloid
dendritic cell influx into the airways in patients with allergic
asthma.\emph{Am J Respir Crit Care Med.} 2008;177(12):1307-1313.

3. Denzler KL, Borchers MT, Crosby JR, et al. Extensive eosinophil
degranulation and peroxidase-mediated oxidation of airway proteins do
not occur in a mouse ovalbumin-challenge model of pulmonary
inflammation. \emph{J Immunol.} 2001;167(3):1672-1682.

4. Malm-Erjef\selectlanguage{ngerman}ält M, Persson CG, Erjefält JS. Degranulation status of
airway tissue eosinophils in mouse models of allergic airway
inflammation. \emph{Am J Respir Cell Mol Biol.} 2001;24(3):352-359.

5. Sur S, Kita H, Gleich GJ, Chenier TC, Hunt LW. Eosinophil recruitment
is associated with IL-5, but not with RANTES, twenty-four hours after
allergen challenge. \emph{J Allergy Clin Immunol.} 1996;97(6):1272-1278.

6. Kikuchi I, Kikuchi S, Kobayashi T, et al. Eosinophil trans-basement
membrane migration induced by interleukin-8 and neutrophils. \emph{Am J
Respir Cell Mol Biol.} 2006;34(6):760-765.

Frauke Prenzler\textsuperscript{1}, Thomas Tschernig\textsuperscript{2},
Katherina Sewald\textsuperscript{1,5}, Tibor Z
Veres\textsuperscript{1,3}, Susanne Rittinghausen\textsuperscript{1},
Norbert Krug\textsuperscript{1,5}, Jens M.
Hohlfeld\textsuperscript{1,4,5}, Armin Braun\textsuperscript{1,4,5}

\textsuperscript{1}Fraunhofer Institute for Toxicology and Experimental
Medicine

\textsuperscript{2}Institute for Cell Biology and Anatomy, Saarland
University, Homburg/Saar, Germany

\textsuperscript{3}Lymphocyte Biology Section, Laboratory of Immune
System Biology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892, USA

\textsuperscript{4} Institute of Immunology, Hannover Medical School,
Hannover, Germany

\textsuperscript{5} Member of the German Center for Lung Research (DZL),
Biomedical Research in Endstage and Obstructive Lung Disease (BREATH)
research network, Hannover, Germany

\textbf{Correspondence:} Armin Braun

Preclinical Pharmacology and Toxicology

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany

email: armin.braun@item.fraunhofer.de

Phone: +49(0)511/5350-263

Acknowledgements: We would like to thank Isabelle Bleeker for processing
the biopsy samples of the classical immunohistology.

Funding: Supported by Deutsche Forschungsgemeinschaft (SFB587/ B8 und
B4)

Conflict of Interest: None of the authors has any financial interest.

Contributions: AB, JH and NK planned and conducted the study. FP, AB and
TT wrote the manuscript, all other authors read, corrected and approved
the manuscript. TZV, KS, SR and FP established and performed the
morphology and made the evaluation of tissue data.

\textbf{Figure legends}

Figure 1: Displayed representative images show 3D-reconstructions of
triple-immunostaining in 100 \selectlanguage{greek}µ\selectlanguage{english}m biopsy sections for
EG2\textsuperscript{+} surfaces as a marker for eosinophils (red
surfaces), NE+ surfaces as marker for neutrophils (yellow surfaces) and
cytokeratin (green surfaces) at baseline (A) and 24 hrs after segmental
allergen provocation (B) of one subject; Settings for image acquiring:
x25-water objective; argon laser (488 nm), helium/neon1 laser (543 nm)
and helium/neon2 laser (633 nm) ; 90 optical slices with an interval of
1.1 \selectlanguage{greek}µ\selectlanguage{english}m; grid spacing: 50 \selectlanguage{greek}µ\selectlanguage{english}m; C-D: Depicted are the graphs of the
quantification of volume of EG+ surfaces {[}\selectlanguage{greek}µ\selectlanguage{english}m/100000 \selectlanguage{greek}µ\selectlanguage{english}m3{]} (C) and
volume of NE\textsuperscript{+} surfaces {[}\selectlanguage{greek}µ\selectlanguage{english}m/100000
\selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{3}{]} (D) before (Baseline) and 24 hrs after
segmental allergen provocation (Allergen). Each symbol represents one
subject (for symbol legend refer to tab. 1). Per subject one 100 \selectlanguage{greek}µ\selectlanguage{english}m
biopsy section comprised of 4 to 7 regions was analysed. The groups were
compared using Wilcoxon matched-pairs signed rank test; **: p
\textless{} 0.01, number of subjects: C: n = 10; D: n = 10; AL = Airway
lumen; EG2 = antibody used to identify ECP; NE= neutrophil elastase; SAP
= segmental allergen provocation.

Figure 2: Images of a granule filled cell positive for EG2 (red)
displayed in the fluorescent channel (A), transmitted light channel (B)
and as a merge of both channels (C); white circle: outline of cell,
white disrupted circle: cell nuclei; D: distribution of
EG2\textsuperscript{+} staining within a tissue volume of a 20 \selectlanguage{greek}µ\selectlanguage{english}m
thickness; E: 3D-reconstruction of the stack image shown in D; white
stars: EG2\textsuperscript{+} staining/surfaces resembling cellular
structure; white small circles: EG2\textsuperscript{+} staining in free
granule structures; thin white arrows: EG2\textsuperscript{+} staining
distributed within the tissue unbound; F -- H: Images show 3D
reconstruction of 100 \selectlanguage{greek}µ\selectlanguage{english}m biopsy sections of three different subjects
24~hrs after segmental allergen provocation. The distribution of
EG2\textsuperscript{+}-staining (red surfaces) beneath the epithelial
layer (green surfaces) is very diverse between the subjects. Settings
for image acquiring A - E: x40-water objective; helium/neon1 laser (543
nm); grid: 10 \selectlanguage{greek}µ\selectlanguage{english}m; scale bar: 10 \selectlanguage{greek}µ\selectlanguage{english}m; F -- H: x25-water objective; argon
laser (488 nm) and helium/neon1 laser (543 nm); 90 optical slices with
an interval of 1.1 \selectlanguage{greek}µ\selectlanguage{english}m; grid spacing: 50 \selectlanguage{greek}µ\selectlanguage{english}m; AL = airway lumen; EG2 =
antibody used to identify ECP; SAP = segmental allergen provocation.

\textbf{Figures}

Figure 1\selectlanguage{english}
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Figure 2\selectlanguage{english}
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