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Cytokine 38 (2007) 145–150

IL-16 signaling specifically induces STAT6 activation through CD4

Changbao Liu, Juliane Mills, Ken Dixon, Joseph Vennarini, Mark Cunningham,
Alfred Del Vecchio, Anuk Das, William Glass *
Centocor Research and Development, Radnor, PA 19087, USA

Received 5 January 2007; received in revised form 18 April 2007; accepted 30 May 2007

Abstract

Biologic activities of IL-16 have been well described (e.g., chemotaxis of CD4+ cells, CD25 upregulation, secretion of IL-1b, IL-4 and
TNF-a secretion) but very few signaling events have been described. To gain a better understanding of how the biologic activities of IL-
16 are regulated following receptor engagement (CD4) we have analyzed the activation state of numerous STAT proteins in primary
human peripheral blood mononuclear cells (PBMCs) and the human monocytic cell line THP-1 following IL-16 stimulation. Of the four
STAT proteins tested, only STAT6 was activated (phosphorylated) in a dose-dependant manner by IL-16. The activation of STAT6 was
completely abolished when IL-16 was pre-incubated with soluble CD4 (the IL-16 cell surface receptor), demonstrating the need for CD4
engagement in STAT6 activation. These results are the first to demonstrate a link between IL-16 and STAT6 activation.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Interleukin-16; STAT6; CD4

1. Introduction stimulation with specific antigen, IL-4, GM-CSF, IL-1b,

IL-16 was first described in 1982 by Cruikshank et al. as
a T cell chemoattractant factor secreted from human
peripheral blood mononuclear cells (PBMCs) [1,2].
Although IL-16 has chemotactic properties, it is not a che-
mokine as it lacks the structural motifs of this family of
proteins [3]. To generate active IL-16, the 631 amino acid
pro-protein is cleaved by caspase-3 to generate a 121 amino
acid protein that has biologic activity [4]. The predominant
cellular source of IL-16 are CD8+ T cells which contain
processed bioactive IL-16 [5–7]. CD8+ cells have been
demonstrated to release 80% of all stored IL-16 within 1–
4 h after a variety of stimuli such as histamine, antigen,
serotonin, GM-CSF, PMA, or C5a [1,2,5,7–9]. Conversely,
numerous other IL-16 secreting cells constitutively generate
pro-IL-16 protein, which requires processing prior to
release. This secretion occurs approximately 12–24 h after
*
Corresponding author. Fax: +1 610 889 4623.
E-mail address: wglass@cntus.jnj.com (W. Glass).
or TGF-b [7–10].
IL-16 exerts its biological effects by binding to domain
D4 of CD4 independently of the TCR complex. IL-16
has a wide array of described biologic activities such as che-
motaxis of CD4+ cells [1,2], upregulation of CD25 [11], IL-
4 and IL-1b expression, forcing cells to enter the G1 phase
of the cell cycle [12,13] and inhibition of proliferation
[14,15].
There has been some investigation into signaling events
that occur after IL-16 engages the CD4 receptor. While
CD4 stimulation in the context if Ag generally results in
p56lck phosphorylation, IL-16 does not induce phosphory-
lation of p56lck but results in only association of p56lck with
the cytoplasmic tail of CD4 [15,16]. Investigations into pro-
tein kinase C (PKC) have demonstrated that IL-16 causes
PKC translocation from the cytosol to the membrane
[17,18]. Other studies have demonstrated the activation of
the p38 Mitogen-Activated Protein Kinase (p38MAPK),
the proto oncogene (c-Jun) and stress-signaling kinase 1
(SEK1), the consequences of which are not known in the
context of IL-16 stimulation [19]. Given that IL-16 causes

1043-4666/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cyto.2007.05.016
146 C. Liu et al. / Cytokine 38 (2007) 145–150
the expression of cytokines and that some cytokine
secretion is dependent on STAT activation, we hypothe-
sized that IL-16 stimulation of CD4+ cells would result
in the activation of one or more STAT proteins. Herein
we describe, for the first time, that IL-16 stimulation of
CD4+ cells results in the specific activation (phosphoryla-
tion) of STAT6, and that this is dependent on the interac-
tion of IL-16 with CD4.
2. Materials and methods
2.1. Soluble human CD4 vector construction
A DNA fragment encoding the mature extracellular
domain of human CD4 (Lys 26–Pro 396) was amplified
from IMAGE Consortium CloneID 5226427 (Open Bio-
systems,Hunstville, AL) using primers 3695 for (5 0 GCG
GCA GCG GCC GCG GCA GCG GCG GCA GCA
AGA AAG TGG TGC TGG GC 3 0 ) and 3695 rev (5 0
CGC GGG CCC TCT AGA TCA TTA TGG CTG
CAC CGG GGT GGA CCA TGT GG 3 0 ). The expression
plasmid, 2759, was created by modifying pcDNA3.1V5-
HIS-TOPO purchased from Invitrogen (Carlsbad, CA) to
include the human growth hormone signal sequence (Met
1–Ala 26, GenBank NP_000506) in frame with the hinge-
CH2-CH3 portions of human IgG1 (Glu 99–Gly 329, Gen-
Bank P01857). The soluble human CD4 PCR fragment and
the expression plasmid, 2759, were ligated together using
NotI and XbaI restriction sites to create plasmid 3695,
which expresses hu IgG1 Fc-fusion on the mature N-termi-
nus of soluble human CD4. Plasmid DNA was prepared
using the Hi-speed Maxiprep kit purchased from Qiagen
(Valencia, CA). Sequences were confirmed using the Big
Dye Terminator Cycle Sequencing Kit purchased from
PE Applied Biosystems (Foster City, CA), followed by
analysis on the Prism ABI377 automated DNA sequencing
apparatus.
2.2. Soluble human CD4 protein expression
Equal amounts of expression plasmid 3695 and pAdvan-
tage (Promega, Madison, WI) were transiently transfected
into human embryonic kidney 293EBNA cells (HEK293E)
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in
serum-containing media. Twenty-four hours after transfec-
tion, the media was replaced with SFMII, (Invitrogen), sup-
plemented with L-glutamine and 5 mM sodium butyrate.
The conditioned media was harvested 5 days after transfec-
tion, 0.8 lM filtered, and kept at 4 C until purification.
2.3. Soluble human CD4 purification
The soluble human CD4-Fc fusion (sCD4-Fc) superna-
tant was loaded onto a IgG affinity column overnight at
1 ml/min at 4 C. The column was then washed with 1·
PBS (Invitrogen) for 10 column volumes (50 ml) at 1 ml/
min. sCD4-Fc was eluted using 100 mM glycine pH 2.5.
Protein was observed by measuring the absorbance at
280 nm. The protein peak was collected and neutralized
in 2 M Tris in a volume of 20% the final elution volume.
The sCD4-Fc sample was dialyzed into 1· PBS overnight
at 4 C. The CD4-Fc sample was filtered through a
0.22 lm filter prior to use. The sample was tested for endo-
toxin by LAL assay and found to be 0.2 EU/mg.
2.4. Cell culture, PBMC preparation, and IL 16 stimulation
THP1 cells (human acute monocytic leukemia cell line,
ATCC 10801, Manassas, VA) were grown in RPMI 1640
with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,
4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate,
and 10% FBS at 37 C in a humidified atmosphere of 5%
CO2. PBMCs were isolated from healthy human peripheral
blood using Ficoll-PaqueTM plus (Amersham Biosciences,
Uppsala, Sweden) density gradient centrifugation. Cells
were washed three times with PBS, and then resuspended
in RPMI-1640 medium (Invitrogen Corporation, Grand
Island, NY) with 10% fetal Bovine serum (Invitrogen Cor-
poration, Grand Island, NY). Prior to Western blotting
and flow cytometric analysis, cells were plated at a density
of 0.5 · 106 cells/per well in a 96-well plate and incubated
with serial dilutions of recombinant human (rh) IL-16
(Fitzgerald Industries International Inc., Concord, MA)
for 20 min at 37 C. For inhibition using CD4, IL-16 was
pre-incubated with CD4-Fc fusion protein or IL-17B R-
Fc fusion protein as a control for Fc (R&D systems) for
30 min prior to inhibition studies. The IL-16 purchased
was stated to have a endotoxin level of 0.1 EU/mg, inde-
pendent assessment by LAL assay shows 0.2 EU/mg (data
not shown). Further, stimulation of cells with this IL-16
resulted in only IL-1b and IL-4 expression, no expression
of IL-6, TNF-a, IFN-g, MIP-1a or MIP1b was seen which
would be expected if endotoxin was present (data not
shown). This material was also confirmed to be IL-16 by
mass spectometry and N-terminal sequencing.
2.5. Western blot analysis
THP1 cells were collected by centrifugation at 20 min
post IL-16 stimulation. Cells were lysed in NuPage LDS
sample buffer with reducing agent (Invitrogen Inc.), then
sonicated, and heated at 70 C for 10 min. The 40 ll of cell
lysate per lane was loaded onto a 4–12% NuPage gel (Invit-
rogen Inc.) and separated by electrophoresis then trans-
ferred onto nitrocellulose membrane. The blots were
probed with anti-STAT and anti-phospho-STAT antibod-
ies (BD Transduction Laboratories, San Diego, CA).
2.6. Flow cytometric analysis
To determine the level of phosphorylated STAT6 pro-
tein, THP1 cells or PBMC, treated or not treated with
IL-16, were collected at 20 min post IL-16 stimulation by
centrifugation. The cells were fixed by incubating cells with
 C. Liu et al. / Cytokine 38 (2007) 145–150 147

Cytofix/Cytoperm buffer (BD Pharmingen, San Diego,
CA) for 10 min at 37 C, then permeabilized by adding cold
Perm buffer III (BD Pharmingen, San Diego, CA) on ice
for 30 min.
The cells were washed twice with staining buffer (1·
PBS, 2% FBS, 0.09% NaN3) then cells were stained with
20 ll of Alexa Fluor-anti-Phosopho-STAT6 (P-STAT6)
and 15 ll of PE-anti-CD4 (BD Transduction Laborato-
ries, San Diego, CA) for flow cytometric (FC) analysis.
Data are presented as percent (%) positive cells. EC50 val-
ues were calculated using GraphPad Prism software (San
Diego, CA).
phorylated form of STAT6. Fig. 2a shows that
unstimulated PBMCs have almost no detectable P-STAT6.
In contrast, PBMCs stimulated with increasing doses of IL-
16 have increasing levels of phosphorylated STAT6. When
dual staining for phospho-STAT6 and CD4 was per-
formed, the cell type responding to IL-16 was the CD4+
population in agreement with the predominance of the lit-
erature (Fig. 2b).
The EC50 of IL-16 inducing phosphorylation of STAT6
was (Fig. 3a) found to be 0.118 lg/ml. To determine the

3. Results

3.1. IL-16 causes STAT6 phosphorylation

Given the lack of information concerning the signaling

events that occur in cells following IL-16 stimulation, we
investigated whether or not IL-16 stimulation of PBMCs
could result in activation of STAT1, 3, 5 or 6. Freshly iso-
lated PBMCs were stimulated with a broad dose range of
IL-16 for 20 min. We then determined the levels of total
and phosphorylated STAT1, 3, 5, and 6 by Western blot.
All STAT proteins investigated were detected in the
PBMCs in a non-activated state (data not shown). Only
STAT6 was phosphorylated in a dose-dependant manner
(Fig. 1). The signal seen with STAT3 was investigated fur-
ther by repeated Western blot at this concentration and by
FACS analysis and determined to be aberrant.

3.2. STAT6 activation by IL-16 in primary and immortalized

cells

To determine if the STAT6 phosphorylation (P-STAT6)

observed by Western blot correlated with active protein in
the cells, FACS analysis was performed with freshly iso-
lated PBMCs. PBMCs were stimulated with recombinant
human IL-16 for 20 min. The cells were then fixed, perme-
abilized and stained with an antibody specific for the phos-

Fig. 2. CD4+ cells respond to rHIL-16 by activating STAT6. PBMCs

Fig. 1. Specific dose-dependent phosphorylation of STAT6 by rHIL-16.
Freshly isolated PBMC’s from normal human donors were cultured at
37 C and treated for 20 min with serial dilutions of rHIL-16 at 37 C.
Equal volume amount of protein were resolved on 4–12% NuPage SDS gel
and transferred onto a nitrocellulose membrane for Western blot analysis.
The blots were probed with mAbs specific for phosphorylated (activated)
STAT1, 3, 5 and 6 (denoted P-STAT). The range of IL-16 used was 2.0 lg/
ml (far left), using 2-fold serial dilutions down to 0.015 lg/ml, with 0.0 lg/
ml being the last column on the right.
were freshly isolated and stimulated with serial dilutions of rHIL-16 for
20 min at 37 C. Cells were fixed, permabilized and stained with (a) a mAb
directed against Phospho-STAT6 or (b) mAbs directed against CD4 and
Phospho-STAT6. (a) The representative histogram presented was gener-
ated by gating on all cells (forward by side scatter). (b) PBMCs were
stimulated as described above and stained with both anti-CD4 mAb (PE)
and anti-Phospho-STAT6 mAb (FITC). The IgG control mAbs show no
staining. The IL-16 (0 lg/ml) shows that these cells are all CD4+ (parental
gate is all cells, daughter gate is CD4+ cells) with none being phospho-
STAT6 positive.
148 C. Liu et al. / Cytokine 38 (2007) 145–150
Fig. 3. Dose–response of IL-16-induced P-STAT6 in PBMCs and THP-1 cells. PBMCs (a) or THP-1 cells (b) were stimulated with different concentrations
of IL-16. AFACS analysis was utilized to measure the number of cells staining positive for P-STAT6. The calculated EC50 for IL-16-induced P-STAT6 in
PBMCs and THP-1 cells is 0.118 lg/ml and 0.860 lg/ml, respectively. Data presented are representative of the three independent experiments.
effect of IL-16 on a cell line, we stimulated a homoge-
neously CD4+ human cell line, THP-1 [20] with a full
dose–response of IL-16 (Fig. 3b). This stimulation resulted
in a dose-dependent increase in STAT6 phosphorylation
further indicating that IL-16 induced phosphorylation of
STAT6 is dependent upon the presence of CD4. The calcu-
lated EC50 using this cell line was determined to be
0.860 lg/ml. This difference may be attributable to differ-
ences in receptor number, signaling machinery, etc.
3.3. IL-16 induced STAT6 phosphorylation is mediated by
CD4
To determine if the STAT6 phosphorylation seen in
response to rhIL-16 was mediated through CD4 (the
described receptor for IL-16), we incubated rhIL-16 with
varying concentrations of soluble CD4 (sCD4-Fc). Fig. 4
demonstrates that soluble CD4 completely inhibited the
activity of rhIL-16. Further, there is a 50% reduction in
the observed stimulation by IL-16 at approximately a 2:1
molar ratio of sCD4Fc to rHIL-16. Incubation of the cells
with the cCD4Fc alone had no effect on STAT6 phosphor-
ylation (data not shown). Further, the use of an irrelevant
Fc-Fusion protein (IL-17B R-Fc) had no effect on the
Fig. 4. IL-16 induced STAT6 activation is blocked with a sCD4-Fc. IL-16
(500 ng/ml) was pre-incubated with a soluble CD4 (sCD4-Fc) or IL-17B
R-Fc fusion (as a control for the Fc) construct for 30 min at 37 C. This
mixture was used to stimulate PBMCs for 20 min at 37 C. Following
stimulation, the cells were fixed, permeabilized and stained with an anti-P-
STAT6 mAb. The IC50 for the soluble CD4 molecule was calculated to be
20 lg/ml. (sCD4-Fc = grey squares, IL-17B R-Fc = black circles).
phosphorylation of STAT6, demonstrating the inhibition
is not a result of the Fc-Fusion. This data, combined with
the above FACS data further indicates that IL-16 binds to
CD4 to initiate STAT6 phosphorylation.
4. Discussion
In the present study we demonstrate that IL-16 can acti-
vate the STAT6 protein through CD4. This conclusion was
drawn from data presented showing that the vast majority
of cells responding to IL-16 were CD4+ cells (although a
small percentage were CD4). This was also demonstrated
by a soluble CD4 molecule completely inhibits STAT6 acti-
vation. The population of cells that are CD4 but still
respond to IL-16 may represent CD9+ cells as this mole-
cule has been previously demonstrated to bind IL-16 [21].
We did not use an anti-CD4 mAb to block the IL-16 activ-
ity on the CD4 receptor as the OKT4 mAb has shown to be
neutralizing but also activating [22]. Additional studies will
need to be conducted to determine if IL-16 is using CD9 to
initiate a signal that subsequently activates STAT6. We
recognize that the dose of IL-16 used to stimulate the cells
is higher than previously published reports that IL-16 is
active at subnanomolar concentrations, however, we were
not able reproduce these findings at the low doses reported.
In our hands IL-16 causes a dose-dependant expression of
IL-1b and IL-4 only when used at concentrations of
>125 ng/ml with an EC50 of 400 ng/ml (data not shown).
These concentrations may seem merely pharmacological
however; they may be physiological in the local tissue
environment.
STAT6 is one of the seven members of the STAT protein
family which, when activated dimerize and rapidly translo-
cate to the nucleus, where they activate or repress the
expression of target genes. Previously, it has been shown
that approximately 35 STAT6-regulated loci have been
described with the large majority being positively regulated
by STAT6 [23]. It has been well described that IL-4 can
induce STAT6 phosphorylation, interestingly the IL-4 locus
also contains a STAT6 binding site [24,25]. As IL-16 stimu-
lation of cells is known to result in IL-4 expression from
 C. Liu et al. / Cytokine 38 (2007) 145–150
CD4+ cells, it is likely that IL-4 expression is a result of
STAT6 activation [24,26]. We investigated IL-4 contamina-
tion in our stock of IL-16 and found no evidence of IL-4 or
of IL-13, the other known STAT6 activator (data not
shown).
It has been shown that stimulating resting CD4+ T cells
with IL-16 results in an intracellular release of calcium.
This calcium release was attributed to the activation (phos-
phorylation) of p56lck [15] . It was later shown that activa-
tion of p56lck was not necessary for all functions of IL-16
but that association of p56lck with the cytoplasmic tail of
CD4 was sufficient to mediate other activities of IL-16
(desensitization of CCR5 and CXCR4) [16,27]. The next
step in this signaling cascade is the activation of phospha-
tidylinositol 1,4,5-trisphosphate, resulting in the increased
expression of the IL-2 receptor [14,28]. Given that the enzy-
matic activity of p56lck was not a necessity, PKC was
investigated and found to play a crucial role in the migra-
tory response of CD4+ cells to IL-16 [17].
One of the predominant pathways mammalian cells use
to convert an extracellular stimulus to an intracellular sig-
nal is by activation of the MAPK subfamilies [29]. Mem-
bers of this family include the extracellular signal-
regulated kinases ERK-1 and ERK-2 [30], the c-Jun-N-ter-
minal/stress-activated protein kinases (JNK/SAPK) and
the subgroups of p38 kinase. IL-16 has been shown to be
a specific activator of the SAPK pathway independent of
the MAPK-family members ERK-1 and ERK-2. Specifi-
cally, stimulation of CD4+ macrophages with IL-16
resulted in phosphorylation of SEK-1, which then activates
the SAPKs p46 and p54, resulting phosphorylation of c-
Jun [19]. In this same study, IL-16-mediated the phosphor-
ylation (activation) of p38 MAPK at Tyr182, resulting in
the activation of the functionally distinct p38 MAPK path-
way. The consequences of these activation pathways was
not elucidated with respect to IL-16 but in other systems,
MAPK activation results in both proliferation and motility
as well as STAT1 activation, indicating this may be the end
result of activating these pathways [31,32]. IL-16 activation
of STAT6 is interesting given that STAT6-deficient mice
show that the STAT6 pathway is the principal signaling
pathway involved in the commitment of CD4+ T cells to
the Th2 phenotype [33].
The identification of IL-16 induced activation of the
STAT6 signaling is an important step towards fully under-
standing the mechanism of action of this cytokine. An
investigation into genes with STAT6 binding sites will pro-
vide further insights into the downstream biological activ-
ities of IL-16.
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