Capsicum ethanol extracts and capsaicin enhance interleukin 2 and interferon gamma production in cultured murine Peyer's patch cells ex vivo

---

ACCEPTED MANUSCRIPT

Capsicum ethanol extracts and capsaicin enhance interleukin-2 and
interferon-gamma production in cultured murine Peyer’s patch cells ex vivo

Fumihide Takano, Masaya Yamaguchi, Satoko Takada, Satoko Shoda, Nobuo Yahagi, Tomoya
Takahashi, Tomihisa Ohta

Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmaceutical
Sciences, Kanazawa University, Kakuma -machi, Kanazawa 920 -1192, Japan




ACCEPTED MANUSCRIPT


Corresponding author: Fumihide TAKANO, Ph.D., Research Associate
Tel: +81-76-234-4470; fax: +81-76-264-6241
E-mail: takano@p.kanazawa-u.ac.jp
1

---

ACCEPTED MANUSCRIPT
Abstract
We investigated the effects of red pepper (Capsicum annuum Lin.) extracts (capsicum extract)
and its main pungent capsaicin on T helper 1 (Th1) and 2 (Th2) cytokine production in cultured
murine Peyer’s patch (PP) cells in vitro and ex vivo. Direct administration of capsicum extract (1 and
10 µg/ml) and capsaicin (3 and 30 µM) resulted in suppression of interleukin (IL)-2, interferon
(IFN)-?, IL-4 and IL-5 production. In an ex vivo experiment using PP cells removed from the mice
after oral administration of capsicum extract (10 mg/kg/day for 4 consecutive days), IL-2, IFN-? and
IL-5 increased in response to concanavalin A (Con A). Oral administration of 3 mg/kg/day capsaicin,
one active constituent of the extract, also enhanced IL -2, INF-? and IL-4 production in response to
Con A stimulation but did not influence the production of IL-5. Orally administered capsazepine (3
mg/kg/day) , a selective transient receptor potential vanilloid 1 (TRPV1) antagonist, slightly enhanced
IL-2 production also irrespective of Con A stimulation. The capsaicin-induced enhancement of both
IL-2 and IFN -? production was not reduced by oral administration of capsazepine (3 mg/kg/day),
suggesting a
ACCEPTED MANUSCRIPT
TRPV1 receptor-independent mechanism. Flow cytometric analysis revealed that the
population of CD3+ cells in the PP cells was significantly reduced while CD19+ cells increased after
oral administration of capsicum extract (1 and 10 mg/kg/day) and capsaicin (0.3 and 3 mg/kg/day).
Capsazepine (3 mg/kg/day) weakly but significantly reversed these effects. Orally administered
capsicum extract and capsaicin did not change the T cell subset (CD4+ and CD8+), Th1 (IFN-?+) and
T2 (IL-4+) ratio. These findings indicate that capsicum extract and capsaicin modulate T cell-immune
responses, and their immunomodulatory effects on murine PP cells are partly due to both
2

---

ACCEPTED MANUSCRIPT
TRPV1-dependent and -independent pathway.

Keywords: Capsic um; capsaicin; Peyer’s patch; Th1/Th2; cytokines; TRPV1
ACCEPTED MANUSCRIPT
3

---

ACCEPTED MANUSCRIPT
Introduction
The inner surface of the intestinal tract possesses a large area of mucosal membranes, which are
continuously exposed to various substances in the intestinal lumen (Mowat et al., 2003) .
Gut-associated lymphoid tissues exist on the intestinal mucosal site and play an important role in the
immune system. Peyer’s patches (PP) are considered to be lymphoid tissues where mucosal immune
responses such as local IgA production and systemic immunological responses are induced (Mowat et
al., 2003). Antigen presentation in PP is important in determining systemic immune responses
including T and B cell-dependent immunity (Mowat et al., 2003; Yoshida et al., 2002). Orally
administrated hot water extract of cultured mycelia of Cordyceps sinensis (Berk.) Sacc was
previously shown to increase interleukin (IL)-6 and granulocyte-colony stimulating factor (GM-CSF)
production by PP cells in mice (Koh et al., 2002). Juzen-taiho-to, a Kampo prescription, was also
shown to enhance production of these cytokines in PP cells from C3H/HeJ mice (Hong et al., 1998).
Moreover, we recently reported that culture filtrate of the medicinal entomogenous fungi
Paecilomyces
ACCEPTED MANUSCRIPT
tenuipes (Peck) Samson (= Isaria japonica Yasuda or Isaria tenuipes) selectively
enhanced T helper 1 cytokine production in cultured murine PP cells from C57BL/6J mice (Takano et
al., 2005), and suggested that oral administration of the culture filtrate might increase immune
responses (Takano et al., 1996), in part due to enhancement of these cytokines. Therefore, to elucidate
the mechanisms of oral immune responses, examination of the cytokine modulating activities in
cultured PP cells will be beneficial.
Capsicum annuum (Solanaceae) is used worldwide not only as a food, due to its pungency, but
4

---

ACCEPTED MANUSCRIPT
also in traditional medicine against gastric ulcers, rheumatism, alopecia and toothache (Szallasi and
Blumberg, 1999). Capsaicin is the most well-known and pungent active ingredient of Capsicum.
Large numbers of studies have established that capsaicin shows various pharmacological effects and
is endowed with a pleiotropic pattern of biological activities, some of which are mediated by the
activation of cellular targets different from vanilloid receptor 1 (Szallasi and Blumberg, 1999).
Capsaicin has also been shown to have immunomodulatory effects, as indicated by its ability to
modulate lymphocyte proliferation and immunoglobulin A, E and G production (Nilsson et al., 1991,
Eglezos et al., 1990), regulate the expression of substance P and its receptor in monocytes (Ho et al.,
1997), and inhibit cytosolic Ca2+ mobilization induced by platelet activating factor in the monocyte
cell lines HL-60 (Choi et al., 2000). However, the systemic T-cell-dependent immune response after
oral administration of capsaicin remains to be clarified.
In this study, we therefore examine the effects of ethanolic extracts of C. annum, capsaicin and a
vanilloid receptor-specific antagonist, capsazepine, on the production of T-helper cytokines in
c
ACCEPTED MANUSCRIPT
ultured PP cells in vitro and ex vivo.

Materials and Methods
Animals
Male C57BL/6J mice, 6 to 10 weeks of age, were purchased from Japan SLC (Shizuoka,
Japan). Mice were housed in groups of five in plastic cages with a 12 h light: 12 h dark cycle and free
access to water and food ad libitum. Adaptation to these conditions for at least 1 week was allowed
5

---

ACCEPTED MANUSCRIPT
before commencing the experiment. The experimental procedures complied with the guidelines of the
Council for Experimental Animals, the Faculty of Pharmaceutical Sciences, Kanazawa University,
Japan. Mice were sacrificed by anesthetization with an overdose of ether.

Materials
RPMI-1640 medium, phosphate -buffered saline (PBS), fetal bovine serum (FBS), penicillin and
streptomycin were obtained from Invitrogen Corp. (Carlsbad, CA USA). Concanavalin A (Con A)
(type IV), type I collagenase, capsaicin and capsazepine were obtained from Sigma Chem. Co. (St.
Louis, MO). All other reagents were purchased from Wako Pure Chemical Co. (Tokyo, Japan). For
analysis of the T, B and T cell subset (CD4+ and CD8+) in PP cells, the following monoclonal
antibodies (mAb, Beckman Coulter Inc., Hialeah, FL) were used: anti-CD45RA-fluorescein
isothiocyanate (FITC) antibodies (RA3-6B2, IgG2a), anti-CD3-FITC antibodies (KT3, IgG2a),
anti-CD4-FITC antibodies (YTS191.1, IgG2b), anti-CD8-phycoerythin (PE) antibodies (KT15,
IgG2a), anti-IFN-?- ACCEPTED MANUSCRIPT
FITC antibodies (XMG1.2, IgG1) and anti-IL-4-FITC antibodies (BVD-24G2,
IgG1). The isotype -matched controls used in this experiment were IgG1 conjugated to FITC, IgG2a
conjugated to PE, IgG2a conjugated to FITC and IgG2b conjugated to FITC.

Extraction and isolation
Capsicum annum L. for medicinal use was purchased from Uchida Wakanyaku Co. Ltd. (Tokyo,
Japan). A voucher specimen of this plant (C05205) was deposited in our laboratory at the Faculty of
6

---

ACCEPTED MANUSCRIPT
Pharmaceutical Sciences, Kanazawa University, Japan.
The dried fruits (1.0 kg) of C. annum were macerated in 95% ethanol (EtOH) (1 L, 3 times) at
room temperature for 24 h. After filtration, the ethanol solution was evaporated under reduced
pressure to give ethanol extract (capsicum extract) (26 g). Capsicum extract (10 g) was then subjected
to column chromatography on silica gel (500 g). Elution with a mixture of hexane-ethylacetate
(AcOEt) and methanol (MeOH) gave 14 fractions: Fr-1 (5% AcOEt, 1200 ml, 698 mg), Fr-2, (25%
AcOEt, 300 ml, 675 mg), Fr-3 (25% AcOEt, 300 ml, 120 mg), Fr-4 (25% AcOEt, 300 ml, 46 mg),
Fr-5 (25% AcOEt, 300 ml, 54 mg), Fr-6 (35% AcOEt, 1200 ml, 458 mg), Fr-7 (50% AcOEt, 600 ml,
78 mg), Fr-8 (50% AcOEt, 600 ml, 210 mg), Fr-9 (65% AcOEt, 1200 ml, 1104 mg), Fr-10 (75%
AcOEt, 600 ml, 119 mg), Fr-11 (75% AcOEt, 600 ml, 80 mg), Fr-12 (90% AcOEt, 600 ml, 141 mg),
Fr-13 (100% AcOEt, 1200 ml, 2844 mg), and Fr-14 (100% MeOH, 1200 ml, 4319 mg). Fr-2 (10 mg)
eluted with 25% AcOEt was determined as being composed mainly of carotenoids and was further
subjected to high performance liquid chromatography (HPLC; ODS, acetone-H2O, 90:10) to give the
main constituent, ?- ACCEPTED MANUSCRIPT
carotene (3.7 mg, Fig. 1). Fr-9 (10 mg) eluted with 65% AcOEt was determined
as being composed mainly of capsaicinoids and was also subjected to HPLC (silica gel,
hexane-acetone, 65:35) to give capsaicin (4.1 mg, Fig. 1). The structures of the carotenoids and
capsaicinoids were determined by comparing their spectral data with those reported in the literature
(Maillard et al., 1997; Mercadante et al., 1999). For ex vivo experiments, capsaicin was purchased
from Sigma Chem. Co. (St. Louis, MO). The purity of the capsaicin was checked and further purified
by HPLC (acetone-H2O, 90:10) before use.
7

---

ACCEPTED MANUSCRIPT

Peyer’s patch (PP) cell preparation
C57BL/6J mice were sacrificed with an overdose of ether and their small intestines were placed
into a petri dish filled with PBS containing penicillin (100 U/ml) and streptomycin (100 µg/ml) on ice
(Manhart, 2001). Visible PPs were carefully dissected out from the wall of the small intestines using
micro scissors under a microscope (10 Peyer’s patches were obtained from each mouse), and were
placed in ice-cold complete RPMI-1640 medium containing 5% FBS, 50 µM 2-mercaptoethanol, 100
U/ml penicillin and 100 µg/ml streptomycin. To obtain a single PP cell suspension, the PPs were
digested with type 1 collagenase (70 U/ml) dissolved in the same medium and incubated for 60 min at
37 oC. After filtration through a 200 µm nylon mesh (Becton Dickinson, Oxnard, CA, USA), the PP
cells were washed three times with complete medium. Cell viability was assessed by trypan blue
exclusion. Morphological analysis by characteristic non-specific esterase and Giemsa staining
revealed that more than 97% of the cells were lymphoids and less than 1% were monocytes. PP cells
(3 × 106
ACCEPTED MANUSCRIPT
cells/ml) suspended in complete medium were seeded in a 24-well tissue culture plate
(Becton Dickinson) and cultured with or without 5 µg/ml Concanavalin A (Con A).(Takano et al.,
2004)

Test sample treatment
Capsicum extracts, fractions, capsaicin and capsazepine were suspended in medium containing
0.3% EtOH solution. For in vitro assay, various concentrations of these test samples (20 µl/well) were
8

---

ACCEPTED MANUSCRIPT
added with or without Con A. For ex vivo assay, capsicum extract (1, 3 and 10 mg/kg/day) or
fractions (3 mg/kg/day) suspended in 10% EtOH and 50 µl/20g body weight solution was orally
injected into mice once a day for four consecutive days. Capsaicin (0.3 and 3 mg/kg/day) or
capsazepine (3 mg/kg/day) (Costa et al., 2004) also suspended in 10% EtOH and 50 µl/20g body
weight solution was orally injected into mice once a day for four consecutive days. In other case, mice
were injected orally with 3 mg/kg/day capsaicin simultaneously with 3 mg/kg/day capsazepine in the
same regimen. Control groups received a vehicle (10% EtOH) instead of test samples. In this
experiment, capsaicin dosage was estimated by capsaicinoids concentration in the effective doses of
both capsicum extract (1 to 2% (w/w) of the extract) (10 mg/kg) and Fr-9 (3 mg/kg) (49 to 51% of the
Fr-9) ex vivo.

Cytokine evaluation
To measure cytokine production in cultured PP cells, culture supernatants were collected at 72 h
and stored at -80 oC ACCEPTED MANUSCRIPT
until use. The levels of IL-2, IL-4, IL-5 and INF-? in the supernatants were
measured by enzyme linked immunosorbent assay (ELISA) using commercial kits (CytoscreenTM,
BIOSOURCE, Camarillo, CA, USA) according to the manufacturer’s instructions.

PP cell viability
To assess the correlation between PP cell cytotoxicity and suppression of cytokine production by
the test samples, PP cells were seeded on a 24-well tissue culture plate (1.5 × 106 cells/0.5 ml/well)
9

---