Shreffler:Basophil Paper

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% \author{Wayne G. Shreffler\corauthref{cor1}\thanksref{label2}}
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\thanks[grant]{This study was supported by AI-079544 and CoFAR}

\title{Protein Tyrosine Phosphatase Inhibition Mimics
Fc$\varepsilon$RI-induced Anergy and Reveals the Immunomodulatory Role of
Hydrogen Peroxide as a Signaling Molecule in Human
Basophils\thanksref{grant}} %Really struggling with a title that captures
this paper -- probably a bad sign!

\author{Caitlin Woo,}
\author{Steven Yoo,}
\author{Winnie Chan,}
\author{Hugh A. Sampson,}
\corauth[ref1]{Reprint Requests: The Immunology Institute and the Jaffe
Food Allergy Institute of Mount Sinai School of Medicine - box 1198, One
Gustave L Levy Place, New York, NY
\author{Wayne G. Shreffler\corauthref{ref1}}
\address{New York, New York}

% keywords here, in the form: keyword \sep keyword
human basophils, desensitization, basophil anergy, syk kinase, Erk, lyn
kinase, protein tyrosine phosphatase, hydrogen peroxide, intracellular
phosphoflow, CD63, CD203c
% PACS codes here, in the form: \PACS code \sep code

\item [-] anergy induction for CD63 upregulation has the same
characteristics reported for the suppression of histamine secretion
\item [-] CD203c is upregulated independently of CD63 and could be useful
as a marker for desensitization
\item [-] Basophil anergy is pathway, but not antigen specific
\item [-] hydrogen peroxide is produced by oxidative burst in basophils
and it can modulate the response to IgE-mediated activation 
\item [-] hydrogen peroxide augments the phosphorylation of Syk, Btk, Erk
by IgE cross-linking
\item [-] degranulation, however, is suppressed (as is 203 upregulation)
-- thought to be via PTPs (orthovanadate also has this effect).
\item [-] A distinct pathway for Erk phosphorylation is activated, and
those cells are anergized for IgE, but not fMLP stim %note here: maybe we
want to do pErk/ 63 staining with fMLP stim
\item [-] while peroxide effect is thought to be secondary to PTPs,
neither lyn, nor syk are activated %should we try to look at fyn?

Word Count = 1701

% ----------------------main text
\section*{Introduction} %-----------------------------------
Successful immunotherapy (IT) of IgE-mediated allergic disease is defined
clinically as a long-lasting diminution of immediate hypersensitivity,
\emph{i.e.}, greater tolerance, that is induced by repeated antigen
exposure over a relatively long time period (\emph{e.g.} weeks-to-months).
In contrast, the clinical term `desensitization' is generally used to
describe a transient hyporesponsiveness that is rapidly induced over hours
by repeated administration of increasing, but -- ideally for the patient --
sub-threshold antigen exposure. The mechanisms of this desensitization or
anergy-induction are thought to be distinct from IT as evidenced by its
contrasting transience and antigen non-specificity.

In settings of IgE-sensitization and ongoing antigen exposure anergy,
hypersensitivity reactions may also be suppressed by effector cell anergy.
For example, patients with clinical tolerance despite readily detectable
specific IgE to food allergens have been observed to experience new onset
hypersensitivity after a period of avoidance.\cite{?} We recently reported
evidence of non-specific suppression of basophils in milk-allergic children
with partial tolerance when they begin to regularly ingest milk

Given new and accumulating evidence for the immunomodulatory role of
effector cells such as
basophils,\cite{AndreaDenzel:2008p1816,Gibbs:2005p1714,Oh:2007p1818} it is
important to better evaluate the nuance of their responses to antigen in
both models of disease as well as human subjects. Suppression of
degranulation by chronic allergen exposure, may or may not be associated
with the suppression of other effector responses, such as leukotriene
synthesis or cytokine production.

Mast cell or basophil anergy, as defined by the suppression of histamine
release after Fc$\varepsilon$RI cross-linking, can be readily induced
\emph{in vitro}, but it is unknown whether the mechanisms of this effector
cell down-regulation are induced during clinical desensitization. In this
study, we tracked surface and intra-celluar events occurring in basophils
at the single-cell level during stimulation via Fc$\varepsilon$RI in order
to evaluate the role of hydrogen peroxide as a second messenger and better
characterize the phenotype of anergized basophils in a manner that could
be applied to future human studies.

\section*{Methods} %-----------------------------------------
%Caitlin -- this is a section you can work on right away and in between
all of your other work. Add text to each section.


\subsection*{Basophil isolation.}

\subsection*{Measurement of oxidative burst.}

\subsection*{In vitro basophil activation and desensitization.}

\subsection*{Measurement of surface and intracellular basophil

\subsection*{Data analysis.}

\section*{Results} % ------------------------------------------------
%------------------------ Define Figures ----------------------------
 \caption{Kinetics and distribution of LAMP (CD107 and CD63) versus CD203c
during basophil activation with anti-IgE.}

 \caption{Effect of extracellular calcium on the upregulation of
activation markers. Solid open histo-grams with calcium, filled histograms
without. Broken line histogram unstimulated.}

 \caption{Basophils are anergized following Fc$\varepsilon$RI
cross-linking. First stim indicated on x-axis. In comparison to PBS
treated control, stim with anti-IgE rendered cells hyporesponsive to a
second round of stimulation (indicated by color, see legend).}

 \caption{Anergy is not antigen-specific. The hapten, NP, induces anergy
in basophils that have been sensitized with anti-NP.}

 \caption{Anti-IgE stimulation of basophils induces reactive oxygen

 \caption{H$_{2}$O$_{2}$ enhances anti-IgE induced phosphorylation of Erk
and Syk. Solid lines indicate median; broken lines represent 25-75th
percentile; n=9.}

 \caption{PTP inhibition at early time points relative to IgE receptor
cross-linking blocks degranulation.}

 \caption{The SFK inhibitor, PP2, blocks H$_{2}$O$_{2}$-induced anergy.}


\subsection*{The kinetics and distribution of IgE-induced LAMPs and CD203c
up-regulation are distinct.} 

We have compared the expression of CD107 (LAMP-1), CD63 and CD203c over
time following anti-IgE stimulation (Figure~\ref{figure1}). The
distribution of both LAMPs is clearly bi-modal and tightly correlated,
consistent with an ‘all-or-nothing’ classical degranulation response.
The peak percent positive response is at 15 minutes and by 120 minutes,
the majority of the cells no longer express high levels of LAMPs on the
plasma membrane. In contrast, the distribution of CD203c remains more
unimodal during activation for most donors, is near maximal by 5 minutes,
and remains elevated at 120 minutes.

The expression of CD63 and CD107 is highly correlated, though CD107
appears to be down-regulated slightly ahead of CD63, resulting in the
emergence of CD63+ CD107dim cells by 45 minutes. CD63+ cells are also
CD203cbright and consistently appear as a subset of cells that have
up-regulated CD203c (Figure~\ref{figure1}).

\subsection*{CD203c up-regulation is not dependent on extracellular

IgE-induced histamine release from basophils is dependent on extracellular
calcium. We compared the sensitivity of CD63 and CD203c up-regulation to
extracellular calcium depletion using EDTA. Consistent with the hypothesis
that CD63 up-regulation corresponds to degranulation, anti-IgE-induced CD63
up-regulation was suppressed by calcium depletion, CD203c up-regulation,
however, was not (Figure~\ref{figure2}).

\subsection*{Fc$\varepsilon$RI cross-linking under calcium free conditions
induces a pathway-specific anergic state in basophils that can be measured
by flow cytometry.}

In vitro induction of anergy in basophils (and mast cells) has been
studied for many years as a method for interrogating the Fc$\varepsilon$RI
signaling pathway and its regulation and for its potential relevance to in
vivo ‘desensitization’. Fc$\varepsilon$RI cross-linking on basophils
in the absence of extracellular calcium, renders them unable to
degranulate upon subsequent stimulation through the same pathway, as
measured by histamine release.\cite{MacGlashan:2008p4410}
We hypothesized that the CD63 response would be similarly suppressed in
anergized cells. Normal donor basophils were isolated ($>$80\% purity) and
stimulated first in the absence of extracellular calcium with anti-human
IgE, washed and then stimulated again in physiological concentrations of
calcium. As expected, in those cells that were first stimulated in the
absence of calcium, there was a dose-dependent suppression of the CD63
response following the secondary stimulus (Figure~\ref{figure3}).

Cells anergized by stimulation via the IgE receptor have been reported to
be suppressed in pathway specific, but non-antigen specific
manner.\cite{MacGlashan:2006p36} To determine whether the same was true of
the CD63 response in our experiments, we also sensitized normal donor
basophils with monoclonal IgE specific for the hapten,
4-hydroxy-3-nitrophenylacetyl (NP). In this way, the small fraction of
unoccupied Fc$\varepsilon$RI receptors of the cell become occupied with
NP-specific IgE, while the majority are occupied with polyclonal donor
IgE. When those cells are stimulated with NP-BSA after mock
desensitization with unconjugated BSA, a small fraction are induced to
upregulate CD63. This sub-optimal stimulation, however, is sufficient to
induce anergy: the median percent CD63 response in nine experiments was
reduced from approximately 20 to 5\% (Figure~\ref{figure4}). 

\subsection*{Basophils produce hydrogen peroxide during Fc$\varepsilon$RI

Nolan and others have shown that hydrogen peroxide is generated during B
cell receptor (BCR) cross-linking and acts as a second messenger to
enhance tyrosine kinase-dependent signaling by specifically and
transiently inhibiting protein tyrosine phosphatases
(PTPs).\cite{Irish:2006p21} Fc$\varepsilon$RI signaling, like BCR,
involves activation of Src family kinases (SFKs) and recruitment of Syk to
phosphorylated ITAMs present in the cytoplasmic tails of receptor complex
membrane proteins. It has been previously reported from cultures of mast
cell lines and basophil-enriched leukocyte preparations that activation by
IgE cross-linking produces reactive oxygen species
(ROS).\cite{Ogasawara:1986p7492} We hypothesized that PTP inhibition may
be important for basophil anergy.

Using the fluorochrome dihydrorhodamine (DHR), we demonstrated that
reactive oxygen species are induced by Fc$\varepsilon$RI cross-linking
(Figure~\ref{figure5}). Increased DHR was detectable at 30 minutes post
stimulation and was strongly inhibited by the superoxide dismutase
inhibitor, diethyldithiocarbamic acid (DETC) and partially inhibited by
N-acetylcystein (NAC).

In order to test the hypothesis that H$_{2}$O$_{2}$ would enhance
signaling, we investigated the effect of exogenous H$_{2}$O$_{2}$ on the
levels of phosphorylated Syk and Erk 1/2. Like Nolan, we found that the
addition of H$_{2}$O$_{2}$ at the time of receptor cross-linking enhanced
downstream phosphorylation of Syk and Erk (Figure~\ref{figure6}).
H$_{2}$O$_{2}$ by itself also modestly induced detectable pErk. Reth has
shown that H$_{2}$O$_{2}$ is sufficient by itself to mimic some effects of
BCR activation.\cite{Reth:2002p23} H$_{2}$O$_{2}$ was unable to induce
degranulation by itself at any concentration tested up to 10 mM (not

\subsection*{PTP inhibition is sufficient to induce anergy.}

Because H$_{2}$O$_{2}$ enhanced phosphorylation of Syk and Erk, we next
hypothesized that H$_{2}$O$_{2}$ would also enhance degranulation. The
effect of H$_{2}$O$_{2}$ on degranulation, however, proved to be dependent
on the timing of its administration relative to receptor cross-linking.
Stimulation with H$_{2}$O$_{2}$ at time points -5, 0, +1 minutes relative
to anti-IgE was inhibitory, while stimulation at +5, and +15 enhanced
degranulation (Figure~\ref{figure7}). The specificity of this effect to
PTPs was suggested by the absence of any suppressive effect on fMLP
signaling (not shown). Treatment of basophils with the irreversible PTP
inhibitor, orthovanadate, also had the same suppressive effect (not
shown). Cell viability was also unaffected by concentrations up to 10 mM
(not shown). Like Fc$\varepsilon$RI cross-linking in calcium free
conditions, peroxide treatment alone induced pathway-specific anergy.
Hydrogen peroxide is a short-lived mediator with a half-life measured in
seconds. We hypothesized that H$_{2}$O$_{2}$-induced anergy was secondary
to a phosphorylation event occurring as a result of transient PTP

\subsection*{H$_{2}$O$_{2}$-induced anergy is dependent on a Src-family
kinase, but not dependent on Syk activation.}

MacGlashan et al. has previously reported that the pathway-specific
anergic state induced by Fc$\varepsilon$RI stimulation is a membrane
proximal event that is not blocked by specific pharmacological inhibition
of PI3K or Syk,\cite{MacGlashan:2008p4410} though it is blocked by SFK
inhibitors, such as PP1 and PP2.\cite{MacGlashan:2000p4438} We found a
dose-dependent inhibition of degranulation as measured by upregulation of
CD63 using inhibitors of SFKs (PP2), Erk (PD98059) and Syk (picetannol)
PI3K (wortmannin); PP2 and wortmannin also blocked CD203c upregulation
(not shown). Neither wortmannin nor picetannol were able to block
H$_{2}$O$_{2}$-induced anergy (not shown), however, PP2 treatment did
prevent anergy induction as predicted from MacGlashan's findings
There are nine mammalian SFKs that have been described. Two members, Lyn
and Fyn, have been repeatedly implicated in basophil activation. We
specifically looked for evidence that PTP inhibition by H$_{2}$O$_{2}$
induced phosphorylation of either of these molecules. Phosphorylation was
detectable with control anti-IgE stimulation, but not with peroxide
treatment (not shown). Other SFKs have been shown to play a role in
Fc$\varepsilon$RI signaling, including Fgr in the mast cell-like line,
RBL-2H3, and Hck in mast cells.\cite{Choi:2004p8317,Hong:2007p8324} It may
be that activation of one or another of these SFKs leads to the anergic
state and identification of that event may represent a specific marker of
anergy that could be used for ex vivo translational studies.

\section*{Discussion} % ------------------------------------------------

Lichtenstein and MacGlashan demonstrated in vitro anergy and have
carefully discriminated antigen-specific and non-specific forms and at
least some of the mechanisms that underly them. Antigen-specific anergy
has been associated with weaker signaling, involving fewer IgE receptor
complexes and transient phosphorylation of syk.\cite{MacGlashan:2003p35} 

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