\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Mast cells derived from systemic mastocytosis exhibit an increased
responsiveness to hyperosmolarity}



\author[1]{Gunnar Nilsson}%
\author[1]{Katarina Lyberg}%
\author[1]{Maria Ekoff}%
\author[1]{Christine Möller Westerberg}%
\author[1]{Camilla Engblom}%
\author[2]{Barbro Dahlen}%
\author[1]{Theo Gulen}%
\affil[1]{Karolinska Institutet Institutionen for medicin Solna}%
\affil[2]{Department of Respiratory Medicine and Allergy Karolinska University Hospital}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
This is a ``Letter to the Editor'' with no abstract.%
\end{abstract}%



\sloppy


\textbf{Mast cells derived from systemic mastocytosis exhibit}

\textbf{an increased responsiveness to hyperosmolarity}

To the Editor,

Systemic mastocytosis (SM) is a disease characterized by increased
number of aberrant mast cells in one or several organs and increased
systemic levels of mast cell mediators \textsuperscript{1}. Indolent SM
(ISM) is the most common form of SM constituting approximately 80\% of
the patients diagnosed with SM. Individuals with ISM often have mediator
mediated symptoms, most commonly from the skin, the gastrointestinal
tract, cardiovascular and respiratory system, but also in the form of
anaphylaxis \textsuperscript{2}. Although basal mediator levels,
including serum tryptase and metabolites of histamine and prostaglandin
D\textsubscript{2} in the urine, are increased at steady
state\textsuperscript{1,3,4}, the symptoms often come as spells without
any obvious trigger suggesting an intrinsic defect causing a
hyper-reactive state of the mast cells, or an endogenous trigger.

We have previously addressed the hypothesis of a hyper-reactive mast
cell phenotype in ISM by \emph{in vivo} provocation of mast cells in the
skin and respiratory tract by skin prick application of morphine and
inhalation of mannitol, respectively \textsuperscript{3}. None of these
triggers mounted a response that was different between ISM patients and
healthy volunteers (HV). To further investigate the hypothesis of a
hyper-reactive mast cell phenotype we also developed mast cells \emph{in
vitro} from 14 ISM patients and 13 HV (same subjects as included
in\textsuperscript{3}). Peripheral blood was obtained, and a
CD34-selection was performed. The CD34-positive progenitor cells were
cultured using a protocol published by Lappalainen et
al.\textsuperscript{5} (for details see supplement). When the cells were
mature, they were plated and exposed to IgE-receptor activation,
morphine or mannitol-induced hyperosmolarity, representing three
distinct activation pathways (see supplement). As a read out for mast
cell activation we measured the release of histamine (as a measurement
of degranulation) and PGD\textsubscript{2} (newly synthesized lipid
mediator); i.e., two prominent mast cell mediators, released through two
different routes, that are increased in ISM.

The growth and development of mast cells \emph{in vitro} from
CD34-selected progenitor cells was followed over a 6-week period. At no
point did we observe any difference between cells from ISM patients or
HV (Figure 1). This result stands in contrast to a study where a
significant increase in mast cell growth from CD34-selected progenitor
cells from ISM patients was described \textsuperscript{6}. An
explanation could be the different culture protocols used in the two
studies but could also be a result of the low numbers of ISM patients
included by Carter et al. (n=4), and the big variation in
growth\textsuperscript{6}. The \emph{in vitro} developed mast cells were
plated and exposed to different mast cells secretagogues: the calcium
ionophore A23187 (1\selectlanguage{greek}µ\selectlanguage{english}M), morphine (1 and 10 \selectlanguage{greek}µ\selectlanguage{english}g/ml), anti-IgE (0.02-20
ng/ml) and mannitol (0.3 and 0.7 M). The release of histamine was
comparable between mast cells derived from ISM and HV in response to all
tested secretagogues (Fig 2A). In contrast, mast cells derived from ISM
showed a significantly increased release of PGD\textsubscript{2} in
response to mannitol, but not to the other tested triggers (Fig. 2B). It
has been reported previously that the release of \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase
(released through degranulation) after IgE-receptor activation is the
same from mast cells derived from ISM as from HV \textsuperscript{6}.
However, in that study they neither investigated the secretion of
PGD\textsubscript{2}, nor other type of secretagogues.

Our study provides the first evidence that mast cells derived from ISM
exhibit an aberrant response profile to mannitol-induced
hyperosmolarity, with no change in degranulation but an increased
synthesis and secretion of PGD\textsubscript{2}, the main eicosanoid
produced by mast cells. A hyper-reactive mast cell phenotype in ISM is
still elusive, but our data indicate that an intrinsic defect in these
cells could affect other signaling pathways than the commonly studied
downstream of the IgE-receptor, and that other mediator releasing
systems than degranulation, i.e., newly synthesized mediators, should be
studied. Finally, the increased levels of PGD\textsubscript{2} in ISM
support the presence of an increased reactivity of their mast cells to
osmolarity changes.

\textbf{Acknowledgement}

We thank Ingrid Delin for technical assistance with the analysis of
PGD\textsubscript{2}. This study was supported by grants from the
Swedish Research Council -- Medicine and Health, the Swedish Cancer
Society, the Swedish Heart Lung foundation, Ellen, Walter and Lennart
Hesselman's foundation, the Konsul TH C Bergh foundation, and through
the regional agreement on medical training and clinical research (ALF)
between Stockholm County Council and the Karolinska Institutet.

\textbf{Conflict of interest}

The authors report no conflict of interest

Katarina Lyberg\textsuperscript{1}, Maria Ekoff\textsuperscript{1},
Christine M\selectlanguage{ngerman}öller Westerberg\textsuperscript{1}, Camilla
Engblom\textsuperscript{1}, Barbro Dahlén\textsuperscript{2}, Theo
Gülen\textsuperscript{1,2}, Gunnar Nilsson\textsuperscript{1,3}

\textsuperscript{1}Division of Immunology and Allergy, Department of
Medicine Solna, Karolinska Institutet, and clinical immunology and
transfusion medicine, Karolinska University Hospital, Stockholm, Sweden

\textsuperscript{2}Department of Respiratory Medicine and Allergy,
Karolinska University Hospital, and Department of Medicine Huddinge,
Karolinska Institutet, Stockholm, Sweden

\textsuperscript{3}Department of Medical Sciences, Uppsala University,
Uppsala, Sweden

\textbf{References}

1. Valent P, Escribano L, Broesby-Olsen S, et al. Proposed diagnostic
algorithm for patients with suspected mastocytosis: a proposal of the
European Competence Network on Mastocytosis.
\emph{Allergy.}2014;69(10):1267---1274.

2. Gulen T, Hagglund H, Dahlen B, Nilsson G. High prevalence of
anaphylaxis in patients with systemic mastocytosis - A single-center
experience. \emph{Clin Exp Allergy.} 2014;44:121-129.

3. Gulen T, Moller Westerberg C, Lyberg K, et al. Assessment of in vivo
mast cell reactivity in patients with systemic mastocytosis. \emph{Clin
Exp Allergy.} 2017;47(7):909-917.

4. Butterfield J, Weiler CR. The Utility of Measuring Urinary
Metabolites of Mast Cell Mediators in Systemic Mastocytosis and Mast
Cell Activation Syndrome. \emph{J Allergy Clin Immunol
Pract.}2020;8(8):2533-2541.

5. Lappalainen J, Lindstedt KA, Kovanen PT. A protocol for generating
high numbers of mature and functional human mast cells from peripheral
blood. \emph{Clin Exp Allergy.} 2007;37(9):1404-1414.

6. Carter MC, Desai A, Komarow HD, et al. A distinct biomolecular
profile identifies monoclonal mast cell disorders in patients with
idiopathic anaphylaxis. \emph{J Allergy Clin
Immunol.}2018;141(1):180-188 e183.



\textbf{Figure legends}

Figure 1. \emph{In vitro} growth and maturation of mast cells over a 70
days period. CD34-positive peripheral blood cells from healthy
volunteers (n= 13) or individuals with indolent systemic mastocytosis
(n=14) were cultured under conditions that promote mast cell
development. Mean ± SEM.

Figure 2. Release of histamine and PGD\textsubscript{2} from
activated\emph{in vitro} developed mast cells. Mast cells were treated
for 30 minutes with calcium ionophore A23187, morphine, anti-IgE or
mannitol and the release of histamine (A) and PGD\textsubscript{2} (B)
was measured in the cell free supernatant. Healthy volunteers (open
boxes) (n=6-10) and systemic mastocytosis (filled boxes) (n=7-13).
Results are shown as box and whiskers; the box extends from the 25th to
75th percentiles and the whiskers min to max. * p\textless{}0.05



\textbf{Figure 1.}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/370969/articles/541176-mast-cells-derived-from-systemic-mastocytosis-exhibit-an-increased-responsiveness-to-hyperosmolarity}



\textbf{Figure 2.}

\textbf{A}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/370969/articles/541176-mast-cells-derived-from-systemic-mastocytosis-exhibit-an-increased-responsiveness-to-hyperosmolarity}

\textbf{B}

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/370969/articles/541176-mast-cells-derived-from-systemic-mastocytosis-exhibit-an-increased-responsiveness-to-hyperosmolarity}

\selectlanguage{english}
\FloatBarrier
\end{document}

