Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 67-72
doi:10.1111/j.1745-7270.2007.00242.x
Differential Expression of Neutrophilic Granule Proteins between Th1
and Th2 Cells
Dee XIE1,2#, Zhiduo LIU1#, Zhenhu LI1,
Yongyong JI1, Jiwu CHEN2, and Bing SUN1*
1 Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Shanghai 200031, China;
2 School of Life Science, East China Normal University,
Shanghai 200062, China
Received: July 14,
2006
Accepted: October
23, 2006
#
These
authors contributed equally to this work
*Corresponding author:
Tel, 86-21-54921376; Fax, 86-21-54921011; E-mail, [email protected]
Abstract T helper cell type 1 (Th1) and 2 (Th2) play central roles in immune
regulation. To identify the novel genes differentially expressed between Th1
and Th2 cells, CD4+ T cells were isolated from DO11.10 transgenic
mice and induced under Th1 or Th2 conditions. Microarray showed differential
expression of neutrophilic granule proteins (NGP) between Th1 and Th2 cells.
NGP was first identified as a myeloid-specific granule protein with homology to
the cystatin superfamily. Here we confirmed greater expression of NGP in Th2
cells by reverse transcription-polymerase chain reaction and real-time
polymerase chain reaction analysis. We also showed that the expression of NGP
mRNA had a peak expression after 5 d culture under Th2- but not Th1-biasing
conditions. Antibody against NGP was prepared, and in concert with the results
of mRNA analysis, the level of NGP protein in Th2 cells detected by Western
blot analysis was also higher than that in Th1 cells. When overexpressed in
HeLa cells, GFP-NGP fusion proteins were localized to the cytoplasm. These
results suggest NGP is a novel marker distinguishing Th2 from Th1 cells and
maybe a novel cytokine secreted by Th2 cells.
Key words neutrophilic granule protein; cystatin; Th2 cell
CD4+ T cells can be divided into T helper cell type
1 (Th1) and T helper cell type 2 (Th2) subsets, which are defined by the
secreted cytokines. Th1 cells secrete interferon-g (INF-g), interleukin
(IL)-2, tumor necrosis factor (TNF)-a and TNF-b, which are critical for
the eradication of intracellular pathogens such as Listeria monocytogenes
and Leishmania major. Th2 cells produce IL-4, IL-13 and IL-5, and they
are necessary for inducing the humoral response to combat parasitic helminthes
and nematodes. The balance between Th1/Th2 subsets determines the
susceptibility to diseases, where the improper development of Th1 cells can
lead to autoimmunity, although an overactive Th2 response can lead to allergy
and asthma [1,2]. Neutrophilic granule protein (NGP) was first identified as a
myeloid-specific granule protein with homology to the cystatin superfamily [3].
Cystatins are natural tight-binding, reversible inhibitors of cysteine protease
[4]. In the immune systems, cystatins can modulate the activity of several
components of the immune response. For example, the allergic lung inflammation
that characterizes a mouse model of human asthma can be inhibited by the use of
an extracellular cysteine protease inhibitor, E64 [5]. Furthermore, parasitic
nematodes act on their hosts immune system by releasing cysteine protease
inhibitors (cystatins) to block effector mechanism [6]. Hartmann et al.
reported that supernatants from Acanthocheilonema rviteae can inhibit CD3-induced
proliferation as a result of the filarial cystatin Av17 [7]. Similarly,
cystatins from Onchocerca volvulus, Nocardia brasilensis and Trichostrongylus
sigmodonitis inhibit CD3-induced proliferation of white blood cells and
splenocytes [8,10]. The effect of cystatins on the immune response is not restricted
to T cell proliferation, because it has been shown that parasitic cystatins
can change the patterns of Th1 and Th2 response in vitro. In vivo,
chronic filarial parasitic infections have also been associated with a switch
to a Th2 response in the host, apparently as a result of filarial cystatin
activity [11,12]. Filarial cystatins can inhibit the hosts response by
inhibiting CD3-induced proliferation, and might have additional roles as modulators
of the secondary immune response [13]. In the present study, we found the higher expression of NGP gene in
Th2 cells than that in Th1 cells by reverse transcription-polymerase chain
reaction (RT-PCR) and real-time PCR analysis. In concert with the results of
mRNA analyses, the level of NGP protein detected in Th2 cells is also
significantly higher than that in Th1 cells. When overexpressed in HeLa
cells, GFP-NGP fusion proteins localized to the cytoplasm.
Materials and Methods
Animals
DO11.10 and BALB/c mice (6–8 weeks old) were purchased from Jackson
Laboratory (Bar Harbor, USA). Mice were kept in a specific pathogen free
facility at Chinese Academy of Sciences (Shanghai, China). Mice care and use
were in compliance with institutional guidelines.
Generation of Th1 and Th2 cells
Naive CD4+ T cells were isolated from DO11.10 spleens
using CD4 T cell subset columns (R&D Systems, Minnesota, USA). The
resulting cells were determined by CD4 staining. Polarized cell
populations were generated by culturing 0.5?106 cells/ml DO11.10 CD4+ T cells
in a 24-well plate with 2.5?106 cells/ml irradiated BALB/c antigen-presenting cells (APCs) (2000
rads) in complete RPMI 1640 with the addition of 2 mg/ml OVA peptide and 10
ng/ml IL-2. For differentiation into Th1 cells, 10 ng/ml IL-12 and 10 mg/ml anti-IL-4
were added to the cultures; for Th2 cells, 10 ng/ml IL-4, 10 mg/ml anti-IL-12
and 1 mg/ml anti-IFN-g were added (all from R&D Systems). The cells were split 1:3 and
fed on day 3. For generation of restimulated cells, Th cells were harvested on
day 7 after primary activation, washed, and stimulated with OVA peptides (2 mg/ml) at 5?105 cells/ml with irradiated BALB/c APCs (2.5?106 cells/ml).
Cytokine measurement
For measurement of cytokines produced by the polarized cells, Th1
or Th2 cells were cultured as described above. Supernatants were collected
after the restimulation for 48 h and assayed using ELISA kit (BD Bioscience,
Bedford, USA).For measurement of cytokines produced by the polarized cells, Th1
or Th2 cells were cultured as described above. Supernatants were collected
after the restimulation for 48 h and assayed using ELISA kit (BD Bioscience,
Bedford, USA).
RT-PCR and real-time PCR
Total RNA was isolated from Th1 and Th2 cells with Trizol reagent
(Invitrogen, Carlsbad, USA) respectively. First-strand cDNA was synthesized
using MMLV reverse transcriptase with 4 mg of total RNA. PCR was carried
out using an aliquot of first-strand cDNA as a template under standard
conditions. For normalization of the amount of RNA loading, RT-PCR of
hypoxanthine phosphoribosyltransferase (HPRT) was performed in each RT-PCR
reaction as an internal control. The primers used for amplification were as
follows: 5‘-CTTTGTATTGGTGGTGGC-3‘ and 5‘-GGTTTCTTGGGTATCCTCT-3‘
for NGP; 5‘-CGAGGTCACAGGAGAA-3‘ and 5‘-TTGGAAGCCCTACAGA-3‘
for IL-4; 5‘-GCCCTTGACTATAATGAG-3‘ and 5‘-GATAAGCGACAATCTACC-3‘
for HPRT.Real-time PCR was performed using SYBR green QPCR master mix
(Applied Biosystems, Foster City, USA) for IL-4, NGP and HPRT. The reactions
were run on the 7900HT fast real-time PCR system (Applied Biosystems). The
thermal cycling conditions were as follows: 50 ?C for 2 min, 95 ?C for 10 min,
followed by two steps PCR for 40 cycles of 94 ?C for 15 s and 60 ?C for 1 min.
Negative control, in which reverse transcriptase was omitted from the reaction,
was run for each sample and reaction for each sample was performed in triplicate.
Data were analyzed according to the relative standard curve methods with
normalizing the values of HPRT expression in each sample. Melting curves for
each PCR reaction were generated to ensure the purification of the amplified
products.
Preparation of rabbit anti-serum and Western blot assay
Each New Zealand white rabbit was immunized subcutaneously at
multiple sites on the back with 0.2–0.8 mg of Escherichia coli-expressed
fusion protein NGP-His in emulsion with complete Freunds adjuvant (1:1, V/V).
After 3 weeks, the rabbits were boosted with the protein emulsified in
Freunds incomplete adjuvant, followed by another intravenous injection of
protein alone in another 3 weeks. Anti-sera were collected 12 d after the last
boost and the immunoreactivity titers were monitored by double agar diffusion
precipitation performed in 0.8% agarose in phosphate-buffered saline (PBS).Protein samples (20 mg) were electrophoresized with 15% sodium
dodecylsulfate-polyacrylamide gel electrophoresis and then transferred onto the
polyvinylidene difluoride membrane with BioRad equipments (Hercules, USA). The
membrane was blocked with 3% bovine serum albumin for 2 h. After incubating
with rabbit anti-sera at 4 ?C overnight, the membrane was stained with
horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, USA)
for 1 h. Then the blotting signals were developed using enhanced
chemiluminescence detection system (Pierce, Rockford, USA).
Confocal microscopy analysis of NGP
The full length NGP gene was amplified by PCR with forward primer 5‘-GGAAGATCTATGGCAGGGCTGTGGAAG-3‘
and reverse primer 5‘-ACGCGTCGACCGGAAATTTTTCAGGATG-3‘ using Th2
cDNA as a template. The fragment was fused in the N-terminal of EGFP coding
region of the expression vector pEGFP-N1. The recombinant pEGFP-N1-NGP or
pEGFP was transfected using Lipofectamine reagent into HeLa cells. The
transfected HeLa cells were incubated with 4‘,6‘-diamidino-2-phenylindole
dihydrochloride for 1 min. Then the samples were washed briefly in PBS before
being observed, mounted onto a microscope slide, and observed by TCS SP2
confocal fluorescence microscopy (Leica, Solms, Germany).
Results
NGP gene is differentially expressed in Th1 and Th2 cells
To discover the novel molecules implicated in Th cell
differentiation, we generated OVA-specific Th1 and Th2 cells from the DO11.10
transgenic mice in vitro and characterized their cytokine secretion by
enzyme-linked immunosorbent assay (ELISA). After restimulation with OVA peptide
for 48 h, the Th1 cells express a relatively high level of IFN-g, whereas the
Th2 cells express a relatively high level of IL-4 (data not shown).After generation of the OVA-specific Th1 or Th2 cells, we used
microarray analysis. The expression of NGP was found to be higher in Th2 than
that in Th1 cells (data not shown). We then confirmed the data by RT-PCR and
real-time PCR analysis. As shown in Fig. 1, there was significantly
higher level of NGP mRNA transcription in Th2 cells than that in Th1 cells. Because the
co-cultures of CD4+ T cells and APC are inevitably contaminated
with granule-containing APC, it could be contributing to NGP expression.
Therefore, we purified naive CD4+ T cells from DO11.10 cells
using fluorescence-activated cell sorting and activated them with plate-bound
anti-CD3 and anti-CD28 under Th1- or Th2-polarizing conditions. For secondary
stimulation, cells were harvested on day 7 after primary stimulation, washed
and restimulated at 1?106 cells/ml with 2 mg/ml plate-bound anti-CD3 and 2 mg/ml anti-CD28 in Th1- or
Th2-inducing culture conditions. We found that NGP expression is induced in
Th1 but not the Th2 pathway [Fig. 1(C)].Furthermore, to examine the kinetics of NGP mRNA expression during
primary stimulation of T cells, CD4+ T cells isolated from
DO11.10 transgenic mice were activated in the presence of Th1- or Th2-inducing
conditions and RNA was prepared on day 0, 2, 3, 4 and 5. In resting naive T
cells, abundant NGP was detected. Furthermore, the expression of NGP gene was
undetectable after the antigen stimulation. Interestingly, on day 5, in T cells
cultured under Th2- but not Th1-inducing conditions, the expression of NGP
gene could be detected again (Fig. 2).
NGP is expressed more in Th2 cells than that in Th1 cells
To determine whether the NGP is highly expressed in Th2 cells, we
expressed the NGP-His fusion proteins in E. coli BL21(DE3) and immunized
the rabbits three times with the purified NGP-His fusion proteins (data not
shown). Then the anti-sera against the NGP-His were confirmed by Western blot
analysis. Fig. 3(A) shows that the anti-sera can recognize the cell
lysates transfected with plasmid pEGFP-N1-NGP but not the control plasmid
pEGFP-N1. In concert with results of the mRNA analyses, Fig. 3(B) shows
that the level of endogenous NGP detected in Th2 cells is significantly higher
than that in Th1 cells. Two distinct bands might be a result of
post-translational modification. This is in line with the result of Moscinski
and Hill [3].
NGP is localized to the cytoplasm
The localization of NGP was monitored by fluorescence confocal
microcopy. In the HeLa cells transfected with pEGFP-N1-NGP, GFP-tagged NGP was
localized in the cytoplasm; but when the cells were transfected with pEGFP-N1,
the GFP protein was dispersed throughout the cells (Fig. 4). Whether it
is localized to the Golgi complex requires further study.
Discussion
The understanding of helper T cell differentiation has increased in
the past several years, but some important questions still remain. We used
microarray to analyze the differential expressed genes between Th1 cells and
Th2 cells and found that NGP was highly expressed in Th2 cells.NGP belongs to the cystatin superfamily. It has been reported that
nematode cystatins were used by the parasitic nematodes to inhibit proteases
involved in antigen processing and presentation, which leads to a reduction of
T cell responses [6,8,9,14]. Similarly, we believe that the host also exists
the similar mechanism to modulate the immune response. In fact, Pierre et
al. showed that cystatin C found in human, rats and mice were involved in
the control of invariant chain degradation and antigen-presentation pathway
[15,16]. Recently, the T-kininogen was found to be able to inhibit
extracellular regulated kinase-dependent T cell proliferation [13]. Human
cystatin salicylic acid (SA) that mainly occurs in salvia and tears [17] can
induce INF-g expression in CD4+ T cells [18]. But whether it
exists the similar proteins in immune systems has not been reported. Here we
showed that the level of NGP was higher in Th2 cell than that in Th1 cells by
RT-PCR and real-time PCR analysis. We further showed that during the
differentiation from naive T cell to effector T cells, the expression of NGP
decreased after activation and increased on day 5 in T cells cultured under
Th2- but not Th1-inducing conditions. Investigation of the amino terminal
sequence of NGP shows two polar amino acids flanking a hydrophobic region,
suggesting a signal sequence and the possibility of post-translational
modification
[3]. The Western blot assay confirmed the level of NGP
protein detected in Th2 cells is also significantly higher than in Th1 cells.
Two distinct bands found might be a result of post-translational modification.
This is in line with sequence analysis. Furthermore, when overexpressed in HeLa
cells, GFP-NGP fusion proteins localize to cytoplasm. These results suggest NGP
might be a novel cytokine secreted by Th2 cells. But whether NGP is associated
with Th differentiation or function as a novel suppressor to inhibit antigen
induced T proliferation need further study.
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