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\begin{document}

\title{Interplay between inflammatory process and platelet activation in
chronic spontaneous urticaria}



\author[1]{Lu Wang}%
\author[2]{Zhiming Lu}%
\affil[1]{Qilu Hospital(Qingdao),Cheeloo College of Medicine,Shandong University}%
\affil[2]{Shandong Provincial Hospital}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
background. Chronic spontaneous urticaria (CSU) is associated with
activation of systemic inflammatory response and platelet activation.To
study whether there is a relationship between inflammatory process and
platelet activation in CSU patients. Methods. Circulating concentrations
of hsCRP ,basophil count and mean platelet volume (MPV) were measured in
patients with CSU of varying severity as well as in healthy subjects,
and we analyzed the relationship between markers and the disease
severity and the correlation between markers. Results. The levels of
hsCRP and MPV were elevated in patients with CSU,and patients with CSU
displayed significantly lower levels of basophil count than the
controls. The levels of hsCRP, basophil count and MPV were associated
with disease severity. A significant association was found between MPV
and hsCRP concentrations in patients with CSU, and basophil count were
respectively correlated with MPV and hsCRP. Conclusions. There may be an
interplay between inflammatory process and platelet activation in the
pathogenesis of CSU.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Chronic spontaneous urticaria (CSU) is a multifactorial mast
cell-dependent inflammatory disease, which is characterized by recurrent
itchy wheals and/or angioedema. Several immunological events have been
described in CSU, which are including inflammatory response and platelet
activation\textsuperscript{1-4}.

It is known, that CRP is produced during an inflammatory reaction and is
elevated in various disorders including infections, cancer, autoimmune,
cardiovascular and gastrointestinal
diseases\textsuperscript{5-7}.Recently, hypersensitive C-reactive
protein (hsCRP) has been shown to be helpful for potential adjunct for
global risk assessment in the primary prevention of cardiovascular
disease\textsuperscript{8}. Moreover,recent findings demonstrated that
basophils may play important roles in a variety of inflammatory skin
diseases\textsuperscript{9}.On the other hand,the platelet parameters
play an impotant and active role in the immune-inflammatory
response\textsuperscript{10,11}. Mean platelet volume (MPV) is an
indicator of platelet size,which has been shown to be the markers of
platelet function and activation\textsuperscript{10}.

In addition,some studies have reported that the evaluation of disease
severity in patients with chronic spontaneous urticaria (CSU) is
essential for the adequate treatment of patients, and higher MPV was
associated with low-grade inflammatory conditions and a variety of risk
factors of cardiovascular disorders\textsuperscript{10,12}.The
correlation between biomarkers may be important in understanding of
pathogenetic processes in CSU and increasing prognostic
value\textsuperscript{2}, yet the prospective studies focusing on these
relationship are still lacking.

Taken together, we sought to see whether there was any correlation
between the markers of inflammatory process and platelet activation in
CSU. Therefore, we measured circulating concentrations of hsCRP,
basophil count and MPV of CSU patients and the healthy subjects, and
then analyzed the relationship between markers and the disease severity
and the correlation between markers.

\textbf{Patients andMethods}

\textbf{Subjects}

Ninety-eight subjects (males : females = 52:46; mean age = 34.5 years;
range 18--70 years) with CSU were evaluated at Department of Dermatology
and selected for the study. The control group consisted of ninty-one
healthy subjects, mean age is 36 years ranging from 20 to 60. All
patients fulfilled the EAACI/GA(2)LEN/EDF/WAO criteria for CSU
(spontaneous appearance of wheals/no obvious external specific trigger
daily, or almost daily for [?] 6~weeks)\textsuperscript{13}. All the
patients showed active urticaria without angioedema at the time of
assessment. None of the examined subjects had taken antihistamines
within at least 4 days before the study, glucocorticoids and
cyclosporine therapy had been withdrawn at least 8~weeks before.None of
these patients were previously treated with omalizumab.Routine
investigations had been performed to exclude concomitant
diseases/conditions that may lead to an increase in systemic
inflammatory response markers,and exclude any known other diseases.

\textbf{Assessment of disease severity}

According to the mean number of wheals which was appeared during a week,
the disease severity was assessed before blood
sampling\textsuperscript{14}: patients with 1 to 10 small wheals
(\textless{}3 cm in diameter) were classified as mild, patients with 10
to 50 small wheals or 1 to 10 large wheals (\textgreater{}3 cm in
diameter) were classified as moderate,and patients with more than 50
small wheals or less than 10 large wheals were classified as severe.

\textbf{Blood collection}

For all measurements, venous blood was collected between 8:00 and 10:00
hours from the antecubital vein in the morning after an overnight fast.
EDTA was used as anticoagulant. The remaining samples were collected and
evaluations were made as part of a standard battery of routine
laboratory examinations in CSU patients.

\textbf{Measurement of whole blood hsCRP,MPV and basophil}

HsCRP concentrations were measured by a immune scattering Nepholometry
method using DELTA specific protein immunoanalyzer(Japan).Basophil count
and MPV concentrations were measured by Sysmex XN-3000 Hematoiogy
System(Japan).All assays were performed according to manufacturer's
detailed instructions.

\textbf{Statistics}

The data in this study were presented as mean \selectlanguage{ngerman}± standard deviation.
Statistical analysis of differences between the two groups was
determined by the Unpaired t test. The correlations were assessed by the
Spearman's rank test.P\textless{}0.05 was considered to demonstrate
statistical differences. The statistical analysis was performed by
graphpad prism (GraphPad Software, San Diego, CA, USA).

\par\null

\textbf{Results}

\textbf{Levels of Whole blood} \textbf{hsCRP ,basophil count and MPV in
patients with CSU}

Concentrations of hsCRP ,basophil count and MPV in patients with CSU
were within their normal ranges. The CSU patients showed significantly
higher concentrations of hsCRP and MPV than the
controls(p\textless{}0.0001;Fig 1 and Table 1). But patients with CSU
displayed significantly lower levels of basophil count than the
controls(p=0.0024;Fig 1 and Table 1).

\textbf{Relationship of clinical} \textbf{disease severity towhole
blood}

\textbf{hsCRP ,basophil count and MPV}

Based on the scale of disease severity, 41 of 98 patients with CSU were
evaluated as severe, 57 as moderate and mild. Patients with severe
urticaria displayed significantly higher levels of hsCRP and MPV than
patients with moderate and mild disease(p\textless{}0.0001; Fig 2 and
Table 2). Patients with severe urticaria displayed significantly lower
levels of basophil count than patients with moderate and mild
disease(p=0.0008;Fig 2 and Table 2).

\textbf{Relationship among levels of whole blood hsCRP ,basophil count
and MPV}

HsCRP were positively correlated with MPV
(r=0.6733,p\textless{}0.0001),there were significant negatively
correlations between basophil count and hsCRP concentrations
(r=-0.6720,p\textless{}0.0001;Fig 3),and MPV were negatively correlated
with basophil count (r=-0.6723,p\textless{}0.0001;Fig 3).

\textbf{Discussion}

In this study, significant differences were found in hsCRP, basophil
count and MPV between CSU patients and the controls,which supports the
assumption that chronic low-grade inflammation and platelet activation
are present in CSU\textsuperscript{15,16}. One possible reason for the
reduced number of basophils in CSU is that these cells are actively
recruited into lesional skin. In the presence of IL-3,chemotaxis is
selectively enhanced in basophils by transendothelial
migration\textsuperscript{17}.The presence of larger platelets in
patients might be a consequence of platelets' consumption at the site of
the urticarial wheals and further compensatory
thrombopoiesis\textsuperscript{18}.

Previous studies found a significant association between CSU severity
and blood levels of CRP\textsuperscript{19}.Ohtsuka T. found that
basopenia and high disease activity may be interlinked.Besides a
significant positive correlation between CU severity score and MPV in
ASST-positive patients was observed by Magen et al.
\textsuperscript{18}. The present findings are consistent with previous
studies.Based on large study it has been indicated that CRP, basophil
count and MPV may serve as biomarkers to monitor CSU severity. In
addition, We found a positive or negative correlation between these
markers, likely reflecting the potential interplay between inflammatory
process and platelet activation in CSU.

It is known that CRP increased due to mast cell activation accompanied
by inflammatory response\textsuperscript{20}. MPV levels correlated with
hsCRP concentration, probably reflecting the same characteristic of the
inflammatory response in CSU.It suggests that there is an interrelation
between the platelets activation and the inflammatory state in the
pathogenesis of CSU. Prior studies have consistently demonstrated that
basophils play a crucial role in IgE-dependent chronic allergic
inflammation in the skin\textsuperscript{21}John A. Eckman et al. have
found altered basophil IgE receptor function in CSU
\textsuperscript{22}. Moreover,some other studies pointed that platelet
activation may occur during immunoglobulin E antibody(1gE)-mediated
reactions. Stimulated basophils release preformed histamine, the major
mediator of urticaria, and newly synthesized factors, such as
leukotriene C4 and interleukin (IL)-4 and IL-13\textsuperscript{23}.
Histamine potentiates platelet activity measured by the aggregation
induced by different agonists, acting through histamine
receptors\textsuperscript{16}. Therefore, we suggest that the underlying
mechanism for this higher activity of platelets may result from local
and/or systemic inflammatory response in CSU.

On the other hand,platelets have been described as a source of
inflammatory mediators that are implicated in histamine release from
basophil and mast cells\textsuperscript{16}.Some studies confirm that
platelet-derived supernatants (PDS) induce histamine release from human
mixed leukocytes containing basophils, one of the initial target cells
in IgE-mediated reactions. Platelets, once activated,can produce a
soluble substance or substances which can initiate basophil-mediated
reactions,further suggesting that platelet activation can enhance
inflammatory reactions\textsuperscript{24}. Interestingly, data from
experimental studies prove that platelet factor-4 (PF-4) fragment is
able to induce rat mast cells to release histamine in a dose-dependent
manner.Moreover, it has been shown that histamine is released by human
platelets in response to aggregatory and immunological stimuli.

Considering the fact that serum histamine-releasing activity is an
important determinant for urticarial severity,it seems that CSU patients
should be intensively treated to ensure comprehensive control of their
symptoms and to reduce inflammatory process and platelet activation.So
it is important to perform repeated blood analyses after symptomatic
treatment and CSU remission. Sequential assessments of HsCRP, basophil
and MPV concentrations, related to severity of CSU might provide
stronger evidence of a causal relationship between platelet activation
and inflammation.Therefore, more research is required to prove such
hypothesis.The analysis of hsCRP, basophil count and MPV is rapid and
inexpensive, therefore combining measurement may be a logical and
practical tool to improve monitoring of CSU severity in patients with
low compliance.

To the best of our knowledge, this study is the \selectlanguage{english}first prospective report
of the correlation among hsCRP,MPV and basophil count. However,
the~limitation~of the~study~was~that biomarkers concentration all remain
within the normal range due to fewer cases. Some improvements may be
brought through increasing the number of patients.

In conclusion,markers of inflammatory process (CRP and basophil count)
and of platelet activation (MPV) are related to each other in CSU
patients, suggesting the possible interaction between these markers in
the disease. There may be an interplay between inflammatory process and
platelet activation in the pathogenesis of CSU.

The correlation between hsCRP,basophil count and MPV would enable
evaluation of the effectiveness of medicines and comparing results of
various clinical or epidemiological studies.In addition,further study is
needed in order to verify the contribution of biomarkers to the
pathological etiology of CSU. Long-term prospective studies are needed
to con\selectlanguage{english}firm whether this also correlates with disease course.

\textbf{References}

1. Zuberbier T, Aberer W, Asero R, et al. The EAACI/GA(2) LEN/EDF/WAO
Guideline for the definition, classification, diagnosis, and management
of urticaria: the 2013 revision and update.
\emph{Allergy.}2014;69(7):868-887.

2. Kolkhir P, Andre F, Church MK, Maurer M, Metz M. Potential blood
biomarkers in chronic spontaneous urticaria. \emph{Clin Exp
Allergy.}2017;47(1):19-36.

3. Triwongwaranat D, Kulthanan K, Chularojanamontri L, Pinkaew S.
Correlation between plasma D-dimer levels and the severity of patients
with chronic urticaria. \emph{Asia Pac Allergy.} 2013;3(2):100-105.

4. Kasperska-Zajac A, Grzanka A, Jarzab J, et al. The association
between platelet count and acute phase response in chronic spontaneous
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5. Vermeire S, Van Assche G, Rutgeerts P. The role of C-reactive protein
as an inflammatory marker in gastrointestinal diseases. \emph{Nat Clin
Pract Gastroenterol Hepatol.} 2005;2(12):580-586.

6. Hage FG. C-reactive protein and hypertension. \emph{J Hum
Hypertens.}2014;28(7):410-415.

7. Kotulska A, Kopec-Medrek M, Grosicka A, Kubicka M, Kucharz EJ.
Correlation between erythrocyte sedimentation rate and C-reactive
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8. Ridker PM. High-sensitivity C-reactive protein: potential adjunct for
global risk assessment in the primary prevention of cardiovascular
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9. Satoh T, Ito Y, Miyagishi C, Yokozeki H. Basophils infiltrate skin
lesions of eosinophilic pustular folliculitis (Ofuji's
disease).\emph{Acta Derm Venereol.} 2011;91(3):371-372.

10. Gasparyan AY, Ayvazyan L, Mikhailidis DP, D.Kitas G. Mean Platelet
Volume: A Link Between Thrombosis and Inflammation? \emph{Current
Pharmaceutical Design.} 2011;17(1):47-58.

11. Kasperska-Zajac A, Rogala B. Platelet function in
anaphylaxis.\emph{J Investig Allergol Clin Immunol.} 2006;16(1):1-4.

12. Chu SG, Becker RC, Berger PB, et al. Mean platelet volume as a
predictor of cardiovascular risk: a systematic review and
meta-analysis.\emph{J Thromb Haemost.} 2010;8(1):148-156.

13. Zuberbier T, Aberer W, Asero R, et al. The EAACI/GA(2)LEN/EDF/WAO
guideline for the definition, classification, diagnosis and management
of urticaria. \emph{Allergy.} 2018;73(7):1393-1414.

14. Sabroe RA, Fiebiger E, Francis DM, et al. Classification of
anti-FcepsilonRI and anti-IgE autoantibodies in chronic idiopathic
urticaria and correlation with disease severity. \emph{J Allergy Clin
Immunol.} 2002;110(3):492-499.

15. Kasperska-Zajac A. Acute-phase response in chronic urticaria.\emph{J
Eur Acad Dermatol Venereol.} 2012;26(6):665-672.

16. Chandrashekar L, Rajappa M, Sundar I, et al. Platelet activation in
chronic urticaria and its correlation with disease
severity.\emph{Platelets.} 2014;25(3):162-165.

17. Motoyasu Iikura, Motohiro Ebisawa, Masao Yamaguchi, et al.
Transendothelial Migration of Human Basophils. \emph{The Journal of
Immunology.} 2004;173:5189-5195.

18. Magen E, Mishal J, Zeldin Y, et al. Increased mean platelet volume
and C-reactive protein levels in patients with chronic urticaria with a
positive autologous serum skin test. \emph{Am J Med
Sci.}2010;339(6):504-508.

19. Kolkhir P, Altrichter S, Hawro T, Maurer M. C-reactive protein is
linked to disease activity, impact, and response to treatment in
patients with chronic spontaneous urticaria.
\emph{Allergy.}2018;73(4):940-948.

20. Kasperska-Zajac A, Sztylc J, Machura E, Jop G. Plasma IL-6
concentration correlates with clinical disease activity and serum
C-reactive protein concentration in chronic urticaria
patients.\emph{Clin Exp Allergy.} 2011;41(10):1386-1391.

21. Mukai K, Matsuoka K, Taya C, et al. Basophils play a critical role
in the development of IgE-mediated chronic allergic inflammation
independently of T cells and mast cells.
\emph{Immunity.}2005;23(2):191-202.

22. Eckman JA, Hamilton RG, Gober LM, Sterba PM, Saini SS. Basophil
phenotypes in chronic idiopathic urticaria in relation to disease
activity and autoantibodies. \emph{J Invest
Dermatol.}2008;128(8):1956-1963.

23. Lourenco FD, Azor MH, Santos JC, et al. Activated status of
basophils in chronic urticaria leads to interleukin-3
hyper-responsiveness and enhancement of histamine release induced by
anti-IgE stimulus. \emph{Br J Dermatol.} 2008;158(5):979-986.

24. Orchard M A, Kagey-Sobotka A, Proud D, M. LL. Basophil histamine
release induced by a substance from stimulated human platelets. \emph{J
Immunol Methods.} 1986(136):2240--2244.

\par\null

\textbf{Tables~}

\textbf{Table 1.} Levels of whole blood hsCRP, basophil count and MPV in
CSU patients and healthy controls\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Patient category\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
No. of patients\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
hsCRP(mg/L)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Basophil count (10\textsuperscript{9}/L)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
MPV (fL)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CSU\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
98\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.750\selectlanguage{ngerman}±1.610\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.028\selectlanguage{ngerman}±0.012\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10.360\selectlanguage{ngerman}±0.776\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
control\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
91\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.465\selectlanguage{ngerman}±1.049\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.033\selectlanguage{ngerman}±0.011\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9.513\selectlanguage{ngerman}±0.672\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p value\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.0024\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Results are expressed as mean \selectlanguage{ngerman}± standard deviation.

HsCRP, hypersensitive C-reactive protein ; MPV\selectlanguage{english},mean platelet volume ;
CSU, chronic spontaneous urticaria.

\textbf{Table 2.} Levels of whole blood hsCRP, basophil count and MPV in
CSU patients with different severity\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
CSU severity & No. of patients & hsCRP(mg/L) & Basophil count
(10\textsuperscript{9}/L) & MPV (fL)\tabularnewline
\midrule
\endhead
Mild/Moderate & 57 & 3.200\selectlanguage{ngerman}±1.310 & 0.031\selectlanguage{ngerman}±0.012 &
10.080\selectlanguage{ngerman}±0.645\tabularnewline
Severe & 41 & 4.515\selectlanguage{ngerman}±1.690 & 0.023\selectlanguage{ngerman}±0.010 & 10.750\selectlanguage{ngerman}±0.780\tabularnewline
p value & - & \textless{}0.0001 & 0.0008 &
\textless{}0.0001\tabularnewline
\bottomrule
\end{longtable}

Results are expressed as mean \selectlanguage{ngerman}± standard deviation.

HsCRP, hypersensitive C-reactive protein ; MPV\selectlanguage{english},mean platelet volume ;
CSU, chronic spontaneous urticaria.

\textbf{Figure legends}

\textbf{Fig 1}

(a) Levels of hsCRP in CSU patients and healthy controls.HsCRP levels
were significantly higher in patients with CSU than in controls

(b) Levels of basophil count in CSU patients and healthy
controls.Basophil count levels were significantly lower in patients with
CSU than in controls

(c) Levels of MPV in CSU patients and healthy controls. MPV levels were
significantly higher in patients with CSU than in controls

\textbf{Fig 2}

\begin{enumerate}
\tightlist
\item
  Levels of hsCRP in patients with CSU divided into two different
  subsets according to the severity of the disease (mild/moderate and
  severe).Patients with severe CSU displayed significantly higher levels
  of hsCRP than patients with mild/moderate CSU
\item
  Levels of basophil count in patients with CSU divided into two
  different subsets according to the severity of the disease
  (mild/moderate and severe). Patients with severe CSU displayed
  significantly lower levels of basophil count than patients with
  mild/moderate CSU
\item
  Levels of MPV in patients with CSU divided into two different subsets
  according to the severity of the disease (mild/moderate and
  severe).Patients with severe CSU displayed significantly higher levels
  of MPV than patients with mild/moderate CSU
\end{enumerate}

\textbf{Fig 3}

\begin{enumerate}
\tightlist
\item
  Relationship between levels of hsCRP,MPV in patients with CSU.A
  significant positively correlation was found between hsCRP and MPV
  levels
\item
  Relationship between levels of basophil count and hsCRP in patients
  with CSU.A significant negatively correlation was found between
  basophil count and hsCRP levels
\item
  Relationship between levels of MPV and basophil count in patients with
  CSU. A significant negatively correlation was found between MPV and
  basophil count levels
\end{enumerate}

Figures

Fig 1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig 2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig 3.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

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

