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Suppressive effects of bee venom on the immune responses in collagen-induced arthritis in rats.

Abstract

The effect of bee venom (BVA) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. Male rats were immunized with an emulsion of 200 [mu]g of CII and complete Freund's adjuvant (CFA). The rats were then given intraperitoneally (i.p.) injection of a suspension of BVA or saline during the experiment. The effect of BVA on cellular responses to CII was examined. In the control rats, the onset of arthritis was observed at the 24th day after the CII-immunization, and the severity of CIA was developed gradually. As compared with rats treated with saline, BVA i.p. injected at doses of more than 20 [mu]l/100 g mouse once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-1 [beta], -2, -6, tumor necrosis factor-[alpha] and interferon- [gamma] when the cells were obtained from rats 24 days after immunization and cultured in vitro with CII. When rats were injected i.p. with sheep red blood cells, hemagglutination titers in BVA-treated and control rats did not differ significantly when low doses of BVA was given to rats. However, i.p. injection of BVA at doses of more than 10 [mu]l/100 g/day suppressed antibody production. Pretreatment of rats with BVA could inhibit the development of collagen arthritis even when 10-20 [mu]/100 g/day of the BVA were used for pretreatment. Interestingly, higher doses than 10 [mu]l BVA/100 g mouse were much effective for arthritis incidence. Treatment of rats with BVA prevented the development of collagen arthritis in a dose-dependent manner. Doses of BVA (15 and 20 [mu] l/100 g) resulted in decreased incidence of arthritis. In conclusion, therapeutic i.p injection with BVA improved the clinical course of the disease and the immune response to CII.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Rheumatoid arthritis; Bee venom; Type II collagen-induced arthritis

Introduction

Bee venom (BVA) (api-toxin) has been widely used in the treatment of some immune-related diseases, especially rheumatoid arthritis (RA) and satisfactory results are obtained (Kwon et al., 2002). Previously, BVA inhibited production of interleukin (IL)-l[beta], tumor necrosis factor-[alpha] (TNF-[alpha]) from macrophages in response to in vivo stimulation with bacterial lipopoly-saccharides when the extract was administered into mice once a day for 7 days (Shin, 2002; Kwon et al, 20012), suggesting that the BVA administered into the patients inhibit cytokine production from both T cells and macrophages and potent effects on RA. It was also investigated the anti-inflammatory effect of the BVA on an acute inflammatory process using the carrageenan-induced edema test (Winter et al., 1962). Treatment with BVA could inhibit the onset and development of arthritis and the immune responses to collagen, but that BVA cannot change the severity when the disease was established. However, little is still known about the mode of action of this toxic medication on RA.

Immunization with type II collagen (CII) is well known to induce inflammatory polyarthritis in rats and susceptible strains of mice (Trentham et al., 1977; Courtenay et al., 1980). Although immune mechanisms that include both humoral and cellular immunity to CII have been implicated in the pathogenesis of the disease (Stuart and Dixon, 1983; Seki et al., 1988), there is much evidence that anti-CII antibodies play an important role in the initiation of the diseases (Kaibara et al., 1985). Since CII-induced arthritis (CIA) in rats and mice is well known to have both clinical and histological similarities to human RA (Trentham et al., 1977; Courtenay et al., 1980), these models has been widely used to evaluate anti-arthritic drugs (Kaibara et al., 1985; Takagishi, et al., 1986). RA is an autoimmune disease in the cartilage and synovial membrane. In the initiation and development of this disease, immunological and inflammatory pathways are critical, and the antigen-specific T cell responses to CII are especially important. Many investigators have tested the hypothesis that the modulation of immune responses to CII, especially the T cell-mediated response, can depress the incidence and the severity of arthritis. Treatments using cytokines and anti-cytokine antibodies have been shown to up-and down-regulate the development of arthritis induced by CII and CFA in rodents (Holmdahl et al., 1990; Assano et al., 1998a, b).

In our previous paper (Kim et al., 2005), we have demonstrated the influence of BVA on osteoblastic cellular responses by using human osteoblastic cells. To evaluate the possible synthesis of estrogen in cells of the osteoclastic lineage, we used the human leukemic cell line FLG 29.1 and the primary first-passage osteoblastic cells (hOB). The results clearly demonstrated that the BVA stimulates armotase activation in FLG19.1 and hOB cells in vitro. For its effectiveness on cellular responses to aromatase expression in the human osteoblastic cells, we also investigated the effects of BVA on the enzyme activity. The FLG 29.1 and hOB cells expressed functional aromatase and BVA induced an increase of both aromatase mRNA and enzymatic activity. However, little is still known about the mode of action of bee venom therapy on RA.

In this paper, we have, therefore, evaluated BVA for its effectiveness on immune responses to CI1 in the rat CIA. In an attempt to gain further insight into the mode of action of BVA, we also investigated the effects of BVA on the incidence and development of arthritis in rat CIA with 2 different regimens: (1) started prior to a primary immunization, (2) started on the day of a primary immunization. The present results show that treatment with BVA, starting concurrently with either the initial or the booster immunization, can inhibit the onset and development of arthritis and the immune responses to collagen, but that BVA cannot change the severity when the disease was established.

Materials and methods

Materials

Rats were purchased from Korea Research Institute of Bioscience and Biotechnology (Daejon, Korea). They were allowed at least 1 week to adapt to the environment (25 [+ or -] 3 [degrees] C, 55 [+ or -] 5% humidity and a 12 h light/dark cycle) and were used at 7 weeks of age. Saline-mixed BVA was obtained from Dongguk University Oriental Medical Hospital (Dr. K.S. Kim) as an i.p. injection grade for human. Each vial contained 10 ml of the BVA. For application in cell culture, randomly selected vials were suspended in normal saline at a concentration of 50 ul/l00 [mu]l. In some cases, original concentration of BVA was used. All other chemicals and biochemicals were of analytical grade and were purchased from Sigma Chem. Co. (St. Louis, MO) or Boehringer Mannheim Biochemicals (Seoul, Korea). Radiochemicals were from Amersham International Co. (Seoul, Korea). All other chemicals and biochemicals were of analytical grade and were purchased from Sigma Chem. Co. (St. Louis, MO) or Boehringer Mannheim Biochemicals (Seoul, Korea). Culture flasks and dishes were obtained from Nunc (Roskilde, Denmark). Unless otherwise stated, all other chemicals were purchased from Sigma (CA, USA). Media and sera for cell culture were purchased from Jeil Biotech Services (Daegu, Korea).

Induction of arthritis in rat

CII collagen (Sigma, St. Louis, MO) extracted from bovine articular cartilage was dissolved overnight at 4[degrees]C in 0.1 M acetic acid at 2.0 mg/ml, after which the solution was emulsified in an equal volume of complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, MI, USA) in an ice-cold water bath. Arthritis was induced by an intradermal injection of 0.1 ml of the cold emulsion into the base of the tail. Rats were boosted subcutaneously with same volume of the emulsion 21 days later. As the CFA control, 0.1 M acetic acid emulsified in an equal volume of CFA alone was injected to control rats using the same schedule. The onset of arthritis was considered to be present when erythema and swelling were detected in at least one joint.

Preparation of lymph mode (LN) cell suspension

Rats were killed under ether anesthesia on day 24 after immunization with CII. Inguinal lymph nodes were removed aseptically and pressed through a 60 gauge steel mesh to give a single cell suspension. After filtering a 200 gauge steel mesh to remove debris and cell clumps, the dispersed cells were washed 3 times with RPMI-1640 medium (BRL Gibco Co., Betheda, MD) containing 10% heat-inactivated fetal calf serum (BRL Gibco), 100 U/ml penicillin, 100 mg/ml streptomycin, 2 x [10.sup.-15] M 2-mercaptoethanol (2ME) and 10mM HEPES, and resuspended in the fresh medium at a concentration of 1 x [10.sup.6] viable cells/ml and used for LN cells.

LN cell culture and culture supernatants

To examine blastic activity of LN cells, 100 [mu]1 to give a total volume of 200 [mu], as described previously (Staines and Wooley, 1994). The mixture was the incubated at 37 [degrees] C in a humidified atmosphere with 5% [CO.sub.2]. After 90 h culture, 37 kBq of (3) H-thymidine ([.sub.3] H-TdR: specific activity 740 GBq/mmol; Amersham Co.) was added to each well, and the plate was maintained for another 6h. Incorporation of (3)H-TdR into cells was measured in a Beckman scintillation spectrometer. To prepare culture supernatants, cells were cultured in 24 well plates at a density of 1 x [10.sup.6] cells in a volume of 1.0ml. Either 50 [mu]g/ml of CII or fetal calf serum (FCS)-free RPMI was added in a volume of 1.0 ml after which the plate was maintained for 48 h at 37 [degrees] C in a humidified atmosphere with 5% [CO.sub.2]. Supernatants were collected after pelleting cells by centrifugation at 100g for 10 min and stored at -40 [degrees] C until used.

Cytokine assays for IL-1[beta], IL-2, IL-6, IFN-[gamma] and TNF-[alpha]

Cytokines of IL-1[beta], IL-2, Il-6, IFN-[gamma] and TNF-[alpha] concentrations in culture supernatants were assayed using rat cytokine ELISA Test kits from R&D Systems (Funakoshi, Co., Ltd., Tokyo, Japan) or BioSource International (CA, USA). Briefly, microplates were coated with 1 [mu]g/ml of anti-cytokine in 50 mM carbonate buffer (pH 9.6) for overnight at 4 [degrees]C, and then the wells were washed 3 times with PBS-0.05% Triton X-100. After blocking with 1% bovine serum albumin (BSA) in phosphor-buffered saline (PBS), samples were added to each well and incubated for 2h at room temperature. The wells were then washed for 5 times. Bound cytokines were detected by biotinated anticytokine and streptoavidin-alkaline phosphatase. After washing, freshly prepared substrate solution (p-nitro-phenol phosphate tablet, Sigma Co.) was added to each well. To stop the reaction of color development, 2 N-NaOH was added to each well after 20 min. OD was measured at 405 nm. The assay was performed in duplicate according to the manufacturer's recommended procedures. The results were expressed as mean [+ or -] SD (pg/ml) of 5 individual rats.

Hemagglutination titers

Rats were injected intraperitoneally with 4 x [10.sup.8] sheep red blood cells (SRBC, Sigma). Scrum hemagglutination titers on day 14 were determined in microtiter plates (Nunc) using microtechnique as described previously (Asano et al., 1998a, b).

Arthritis assessment

The clinical symptoms of arthritis in all 4 limbs were evaluated with a visual scoring system. Arthritic lesion of a scale of 0-4: 0 = no change, 1 = swelling and erythema of the digit, 2 = mild swelling and erythma of the limb 3 = gross swelling and erythema of the limb, 4 = gross deformity and inability to use the limb. The arthritis score of each rat was the sum of the scores of each of the 4 limbs, the maximum score being 16. A rat that showed a score of 1 or more was regarded to be arthritic. The incidence and day of onset of arthritis were also recorded.

Measurement of delayed-type hypersensitivity (DTH)

DTH to CII was assessed by the rat ear skin test according to the method described by Cremer et al. (1983). The changes in ear thickness in mm at 48 h were measured after intradermal injection with 10 [mu]g CII dissolved in 10 [mu]l 50 mM Tris-HCl buffer (pH 7.2). The opposite ear was injected with an equal volume of 50 mM Tris-HCl buffer and served as a control. Measurement was made with a dial thickness gauge (Ozaki Sangyou, Tokyo, Japan) and the results were expressed as the difference in thickness between collagen-and buffer-injected ears. Also, the percentage of swelling is calculated by the following formula; (the thickness of footpad after boosting)/(the thickness of foodpad before the boosting) x 100 (%).

Analytical methods

Protein contents were determined by a Protein assay kit of Bio-Rad Laboratories (Richmond, CA, USA). Phospholipase A2 (PLA2) isolation has been isolated from crude bee venom using a highly efficient reverse phase high performance liquid chromatographic method, as described previously (Ameratunga et al., 1995). Melittin, apamin, MCD peptide, histamine and hyaluronidase have been determined as described previously (Lariviere and Melzack, 1996; Gevod and Birdi, 1984).

Statistics

Results were expressed as means [+ or -] SE. Differences were evaluated for significance with the nonparametric Dunnett's multiple comparison test for the arthritic scores, with the Cox-Mantel test for the incidence of arthritis, and the significance of difference between the two groups were evaluated by Student's t test.

Results

Component of bee venom

Components of saline-mixed BVA have been analyzed. As shown in Table 1, BVA contains a variety of peptides including melittin, apamin, and the mast-cell-degranulating (MCD) peptide. It also contains enzymes such as phospholipase A2 (PLA2) and hyaluronidase. In addition, a small amount of a biologically active amine, histamine, was included in BVA (Table 1).
Table 1. Component of bee venom used in this study

Components Contents (% of dry BV)

Small peptide 44.4
Melittin 40.0
Apamin 2.0
Histamin 0.1
MCD-Peptide 401 2.0
Hyaluronidase 1.5
Phospholipase A2 10.0


Influence of BVA on proliferative response of lymphnode cells from rats immunized with collagen type II

The study was designed to examine the effect of BVA on cellular immune responses to CII. Rats were treated i.p. with various doses of the extracts for 14 days from obtained 21 days after immunization and cultured with or without 50[mu]g/ml of CII. The result are shown in Table 1. LN cells prepared from non-immunized rats did not respond to in vitro stimulation with CII as measured by [.sup.3]H-TdR incorporation. However, cells prepared from immunized rats proliferated extensively when cultured in the presence of CII.

[.sub.3] H-TdR incorporated into the cells when medium was used with a dose of 10[mu]l/100g mouse, a value of 2.9 [[+ or -][+ or -]or--] 0.37 (x[l0.sup.3]) was measured. However, CII group showed 67.4 [+ or -] 9.6 ( x [10.sup.3]) H-TdR incorporation into the cells. Also, [.sub.3] H-TdR incorporated into the cells when medium was used with a dose of 15 [mu]1/100 g mouse, a value of 3.1[+ or -]0.42 (x[10.sup.3]) was measured. However, CII group showed 65.4[+ or -]7.6 [( x [10.sup.3]).sup.3] H-TdR incorporation into the cells. In contrast, when [.sub.3] H-TdR incorporation was measured with a dose of 20 and 30 [mu]l/100 g mouse, the values was observed to be 2.5 [+ or -] 0.54 (x[10.sup.3]) and 2.1[+ or -]0.19 (x[10.sup.3]), respectively. CII group showed 9.8[+ or -]1.3 (x[l0.sup.3]) and 8.3 [+ or -] 1.2 of [.sub.3] H-TdR incorporation. These results indicate that treatment of rats with bee venom (api-toxin)-aqua-acupuncture at doses of 10[mu]l00g (3[mu]g/30g mouse) and 15[mu]l/100g (5 [mu]l/30 g mouse) scarcely affected the proliferative response of LN cells and showed the similar levels of [.sub.3] H-TdR uptake to that of immunized, saline-treated rats.

Effect of BVA on cytokine production from lymph node cells in response to CII stimulation in vitro

Continuous i.p. injection (1 time per 2 days) of BVA at doses of more than 20 [mu]l/100 g dramatically inhibits the proliferative response to in vitro stimulation of CII; the levels of [.sub.3] H-TdR incorporation in experimental rats were identical to that in non-immunized controls. The effect of BVA treatment on cytokine production from LN cells in response to CII stimulation was examined in the next experiments. As shown in Tables 2 6, LN cells prepared from immunized rats could secrete much higher levels of T cell cytokines IL-1[beta] (Table 2), IL-2 (Table 3), IL-6 (Table 4), TNF-a (Table 5) and IFN-[gamma] (Table 6) in response to CII stimulation. Although treatment of mice with low doses of BVA (10 and 15[mu]l/ l00g) did not suppress the ability of LN cells to secrete T cell cytokines, i.p. injection of BVA at doses of more than 20[mu]l/100g significantly suppressed the production of those cytokines of IL-1[beta], IL-2, IL-6, TNF-[alpha] and IFN-[gamma] from LN cells.
Table 2. Effect of BVA on proliferative response of lymph node cells
from rats immunized with collagen type II

[.sup.3] H-TdR Dose of BVA ([mu]l/100 g mouse)
incorporated
(x [10.sup.3])

 0 5 10

Medium 3.3 [+ or -] 3.2 [+ or -] 0.43 2.9 [+ or -] 0.37
 0.32

CII 78.5 [+ or -] 76.8 [+ or -] 9.9 67.4 [+ or -] 9.6
 9.6

[.sup.3] H-TdR Dose of BVA ([mu]l/100 g mouse)
incorporated
(x [10.sup.3])

 15 20 40

Medium 3.1 [+ or -] 2.5 [+ or -] 0.54 2.1 [+ or -] 0.19
 0.42

CII 65.4 [+ or -] 9.8 [+ or -] 1.3 ** 8.3 [+ or -] 1.2 **
 7.6

Lymph node cells were obtained from rats 24 days after immunization
with CII. The cells were the cultured in triplicate in the presence or
absence of 50 [mu]g/ml CII for 96 h. The proliferation of cells was
assayed by addition of 1.0 [mu]Ci (3) H-thymidine for the final 6 h in
culture. The data was expressed as mean + SD (in counts per minute) of
5 individual mice.

** p < 0.001 (significant compared to control).

Table 3. Effect of BVA on IL-l[beta] production from LN cells in
response to CII stimulation in vitro

IL-1[beta] Dose of BVA ([micro]l/100g)

 0 5 10 15 20 40

Medium 5.9 9.2 8.9 9.3 6.5 5.6
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 1.5 0.9 1.1 1.2 0.5 0.7

CII 164.5 176.8 197.4 19.4 17.8 8.9
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 19.5 29.4 29.8 2.4 ** 2.5 ** 119 **

Lymph node cells were obtained from rats 24 days after immunization
with CII. Supernatants were prepared 48 h after culture and cytokine
concentration was measured by ELISA. The data were expressed as mean +
SD (picogram per ml) of 5 individual mice.

** p < 0.001 (significant compared to control).

Table 4. Effect of BVA on IL-2 production from lymph node cells in
response to CII stimulation in vitro

IL-2 Dose of BVA ([micro]l/100g)

 0 5 10 15 20 40

Medium 1.2 4.5 3.8 4.1 2.8 2.5
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 1.0 0.5 1.2 1.3 0.9 0.7

CII 554.9 595.2 600.9 9.7 8.6 8.9
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 55 65 69 1.8 ** 1.5 ** 2.1 **

Lymph node cells were obtained from rats 24 days after immunization
with CII. Supernatants were prepared 48h after culture and cytokine
concentration was measured by ELISA. The data were expressed as mean
[+ or -]SD (picogram per ml) of 5 individual mice.

** p < 0.001 (significant compared to control).

Table 5. Effect of BVA on 1L-6 production from lymph node cells in
response to CII stimulation in vitro

IL-6 Dose of BVA ([mu]l/100 g mouse)

 0 5 10 15 20 40

Medium 123.9 145.2 138.9 129.3 132.5 153.6
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 1.5 0.9 1.1 1.2 0.5 0.7

CII 523.4 575.4 538.5 529.6 282.3 165.3
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 65 87 64 67 45 ** 43 **

Lymph node cells were obtained from rats 24 days after immunization
with CII. Supernatants were prepared 48 h after culture and cytokine
concentration was measured by ELISA. The data were expressed as mean
[+ or -] SD (picogram per ml) of 5 individual mice.

** p < 0.001 (significant compared to control).

Table 6. Effect of BVA on TNP-a production from lymph node cells in
response to CII stimulation in vitro

TNF- Dose of BVA ([micro]l/100g)
[alpha]

 0 5 10 15 20 40

Medium 165.1 176.5 168.5 159.7 123-3 98.5
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 15 29 21 22 18 14

CII 874.1 921.3 897 619.8 217.4 128.5
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 165 154 98 98 ** 34 ** 45 **

Lymph node cells were obtained from rats 24 days after immunization
with CII Supernatants were prepared 48h after culture and cytokine
concentration was measured by ELISA. The data expressed as mean
[+ OR -] SD (picogram per ml) of 5 individual mice.
** p<0.001 (significant compared to control).


Effect of BVA on hemagglutination titers to sheep red blood cells (SRBC)

The next experiments were carried out to examine the effect of BVA on antibody production. Rats were injected intraperitoneally with SRBC, and serum was obtained 14 days later to examine hemagglutination titers. Hemagglutination titers in BVA-treated and control rats did not differ significantly when low doses of the BVA were given to rats (Table 7). However, i.p. injection of BVA at doses of more than l0 [mu]l/l00g/day for 14 days slightly suppressed antibody production, as shown to be 7.8 [+ or -] 0.8 HA titers, while i.p. injection of BVA at closes of more than 15-20 [mu]1/100 g/day for 14 days markedly suppressed antibody production, as observed to be 3.7 [+ or -] 0.04 and 2.9 [+ or -] 0.6 liters (Table 7).
Table 7. Effect of BVA of IFN-[gamma] production from lymph node cells
in response to CII stimulation in vitro

IFN- Dose of BVA ([mu]l/100 g mouse)
[gamma]

 0 5 10 15 20 40

Medium 1.6 3.8 2.9 2.4 1.4 0.6
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 1.4 1.3 0.9 0.5 0.7 0.9

CII 954.9 1205.6 1106.4 998.7 56.7 45.9
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 87 187 154 134 87 ** 67 **

Lymph node cells were obtained from rats 24 days after immunization
with CII. Supernatant were prepared 48h after culture and cytokine
concentration was measured by ELISA. The data were expressed as mean
[+ OR -] SD (picogram per ml) of 5 individual mice.
** p < 0.001 (significant compared to control).


Effect of BVA before the primary immunization with CII

Rats were i.p. injected with various doses of BVA on days 12 and 0 relative to the primary immunization with CII. Pretreatment of rats with BVA could inhibit the development of collagen arthritis even when 10-20 [mu]1/100 g/day of the BVA was used for pretreatment (Table 8). When doses of 5 and 10 [mu]1 BVA/100 g mouse were injected, arthritis incidence showed 6/6 and 4/6, respectively, indicating that only 10 [mu]l BVA/l00 g are effective for arthritis. Interestingly, higher doses than 15 and 20 [mu]l BVA/l00 g mouse were much more effective for arthritis incidence, showing each of 1/5 and 1/4. Similar results were observed in parameters of Arthritis index, days of arthritis onset, antibody levels (24 and 48 days) and DTH.
Table 8. Effect of BVA on bemagglutination titers to sheep red blood
cells (SRBC)

 Dose of BVA ([mu]l/100 g mouse)

 0 5 10 15 20 40

HA 10.5 13.2 7.8 3.7 2.9 1.5
titer [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 1.0 1.1 0.8 * 0.4 ** 0.6 ** 0.6 **

Rats (5/group) were injected intraperitoneally with 4 x [10.sup.8]
crythrocytes on day 0. Various doses of BVA were i.p. injected into
rats for 14 consecutive days staring on the day of immunization.
Hemagglutination titers (HA) were measured on day 14 and the result
were expressed as mean [+ or -] SD.
* p < 0.01, **p < 0.001 (significant compared to control).


Effect of BVA treatment from the day of the primary immunization with CII

Rats were 14 daily i p. injected with various dose of BVA or saline starling on the day of the primary immunization with CII. Treatment of rats with BVA prevented the development of collagen arthritis in a dose-dependent manner (Table 9). A satisfactory significant prevention of the disease was achieved by treating the rats with 20)11/100 g/day of the BVA, while no clear effects were produced by the treatment with lower dose (10 [mu]l/100 g) of BVA. The effects were accompanied by the inhibition of DTH to collagen measured on Day 48 and of anti-CII antibody production on Days 24 and 48. Doses of BVA (15 and 20 [mu]l/100 g) resulted in incidence of arthritis of 2/5 and 1/6, respectively. Arthritis indexes of the two doses showed 5.2 [+ or -] 0.6 and 3.1 [+ or -] 0.2, respectively. On the other hand, days of arthritis onset were 31.2 [+ or -] 3.5 and 30.3 [+ or -] 2.1, respectively. Similar results were also observed in antibody levels and DTH of the two doses to be 6.7 [+ or -] 0.2 and 2.3 [+ or -] 0.5 (for 24 days), 33.4 [+ or -] 3.5 and 13.2 [+ or -] 1.3 (for 48 days) and DTH of 45.3 [+ or -] 6.5 and 12.7 [+ or -] 3.2 (for 24 days), respectively (Table 10).
Table 9. Effect of BVA treatment (days 12 to 0) on the development of
collagen arthritis

 Dose of BVA ([micro]l/100g mouse)

 5 10 15 20 Saline

Incidence of 6/6 4/6 1/5 1/4 9/9
arthritis

Arthrilix 13.2 12.9 10.3 10.1 19.6
index [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 l.l 1.3 1.4 * 1.4 * 1.1

Days of 30.5 30.6 32.1 32.0 29.2
onset [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 3.5 4.1 3.0 0.25 2.3

Antibody
levels
(mg/ml)

 24 days 24.8 28.6 29.5 29.7 30.2
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 7.6 4.5 5.4 3.3 6.1

 48 days 129.8 126.4 123.5 123.2 130
 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 13.8 15.1 21.0 11.4 14.5

 DTH 73.3 72.8 69.4 65.4 74.2
 ([10.sup.-2] [+ or -] [+ or -] [+ or -] [+ or -] [+ or -]
 mm) 6.1 10.6 7.1 8.1 9.9

The results except for "arthritis incidence" were expressed as mean
[+ or -] SD.

* p < 0.05; ** p < 0.001 (significant compared to control).

Table 10. Effect of BAV treatment (days 0-14) on the development of
collagen arthritis

 Dose of BVA ([mu]l/100 g)

 10 15 20 Saline

Incidence of 6/6 2/5 1/6 9/9
arthritis

Arthritix index 10.3 [+ or -] 5.2 [+ or -] 3.1 10.5
 2.3 0.6 * [+ or -] [+ or -]
 0.2 1.2

Days of onset 30.6 [+ or -] 31.2 [+ or -] 30.3 30.5
 4.4 3.5 [+ or -] [+ or -]
 2.1 3.2

Antibody levels
(mg/ml)

 24 days 23.2 [+ or -] 6.7 [+ or -] 2.3 24.4
 2.9 0.2 ** [+ or -] [+ or -]
 0.5 2.4

 48 days 111.4 [+ or -] 33.4 [+ or -] 13.2 123.3
 12.2 3.5 ** [+ or -] [+ or -]
 1.3 12.3

 DTH (10-2mm) 63.2 [+ or -] 45.3 [+ or -] 12.7 69.4
 7.6 6.5 * [+ or -] [+ or -]
 3.2 ** 7.6

The results except for "arthritis incidence" were expressed as mean
[+ or -] SD.
* p < 0.05; ** p < 0.001 (significant compared to control).


Discussion

BVA is widely used in the chronic management and the treatment of RA, particularly, in Korea (Kwon et al., 2001, 2002). However, the mechanism by which the understood. BVA inhibited production of IL-1[beta] and TNF-[alpha] from macrophages in response to in vivo stimulation with bacterial lipopolysaccharides when the extract was administered into mice once a day for 7 days (Shin, 2002; Kwon et al., 2001, 2002), suggesting that the BVA administered into the patients inhibit cytokine production from both T cells and macrophages and potent effects on RA. BVA treatment, which began concurrently with a booster injection, also significantly suppressed the development of arthritis and immune responses to collagen. The precise mechanisms accounting for these phenomena are not clear, but similar observations were made by Asano et al. (1998a, b), who showed that delayed traditional Chinese extract treatment could suppress development of arthritis and of immunity to collagen. It is observed that BVA is able to suppress clonal expansion of helper T cells, when it is administered intraperitoneally into rat. Therefore, although the mechanism(s) by which BVA exerts suppressive effects on clonal T cell expansion is not well understood, this regimen might theoretically lead to specific clonal depletion and results in inhibition of development of the diseases. An alternative explanation is that the time of a booster injection may still be within the induction phase of arthritis.

In this study, we examined the influence of BVA on cellular immune responses by using rat CIA, an experimental model for RA. The present results clearly demonstrated that the BVA strongly inhibits T cell activation including blastogenesis and cytokine production in response to antigenic stimulation in vitro. We examined the effect of the i.p. injection of BVA on the development of CIA in rats and on immune responses to CII. We observed that the BVA injection has significant reductive effects on the development of CIA in rats at dosages of 100-20 [mu]l/100g/week. The excessive production of several cytokines such as IFN-[gamma], IL-1[beta] and TNF-[alpha] in the locally affected joints are also observed in these two diseases (Mauri et al., 1996; Gattorno et al., 1997). IFN-[gamma] is known to play an important role in the pathogenesis of RA by inducing and enhancing the expression of class of RA by inducing and enhancing the expression of class II MHC antigens (Nakajima et al., 1990). IL-1[beta] and TNF-[alpha] are also thought to be involved in cartilage destruction by stimulating the synthesis of metallproteinase (Dayer et al., 1985; Schnyder et al., 1987) and by inhibiting proteoglycan synthesis (Tyler, 1985). In our present study, it is speculated that BVA inhibits immune responses, especially cytokine production and antibody formation, and helps to modify the clinical condition of the patient with RA. However, drawing the conclusion that inhibitory action of BVA on immune responses is responsible for beneficial effect of the BVA on RA, it is necessary to examine the following questions: (1) whether the immune suppressive activity of BVA might contribute to the therapeutic mode of action of the BVA on RA and (2) if observed, when the most effective times of starting the treatment with BVA are. Therefore, we have used rat CIA and examined a second part of experiment to answer the questions described above.

It is reported that immunization with CII did not induce arthritis or immunity to collagen in congenitally athymic nude rats (Takagishi et al., 1986). Since the clinical treatment with immunosuppressive agents such as cyclosporin A, FK-506 and medicinal plants had a beneficial effect in patients with refractory RA (Weinblatt et al., 1987; Yocum, 1994; Asano et al., 1997; Yang et al., 1992), BVA might be a useful tool for the treatmetn of RA. The recommended dose of BVA in the management and treatment of rat CIA will be 20 [mu]l/100 g, which is two-fifth of the human therapeutic dose. However, biochemical and metabolic analysis of the constituents of BVA have to be analyzed in further delineating its mechanisms of action in arthritis. The results explain that BVA is effective for the treatment of osteoarthritis in patients.

Acknowledgment

This study was in part supported by the Biogreen 21 Grant, Rural Administration, MOA, Korea.

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Kyung-Woon Kim (a), Yong-Seung Shin (a), (b), Kap-Sung Kim (b), Young-Chae Chang (c), Kwan-Kyu Park (c), Jae-Bok Park (c), Jung-Yoon Choe (c), Kwang-Gill Lee (d), Mi-Suk Kang (e), Young-Guk Park (f), Cheorl-Ho Kim (a), *

(a) Molecular and Cellular Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, Chunchun-Dong 300, Suwon, Kyunggi-Do 440-746, Republic of Korea

(b) Department of Oriental Medicine, Dongguk University, Kyungju 780-714, Republic of Korea

(c) Department of Pathology and Medicine, Catholic University of Daegu Medical School, Daegu, Republic of Korea

(d) Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Republic of Korea

(e) Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyungwon University, Kyunggi-Do 461-701, Republic of Korea

(f) School of Dental Medicine, Kyung Hee University, Dongdaemun-Gu, Republic of Korea

Abbreviations: R.A. rheumatoid arthritis; BVA, bee venom: CII, type II collagen; CIA, collagen-induced arthritis; CFA, complete Freund's adjuvant; i.p.; intraperitoneally; IL, interleukin; TNK, tumore necrosis factor; hOB, osteoblastic cells; 2ME, 2-mercaptoethanol; LN, lymph node; FCS, fetal calf serum; BSA, bovine serum albumin; PBS, phospho-buffered saline: SRBC, sheep red blood cells; DTH, delayed-type hypersensitivity.

* Corresponding author. Tel.: +8231 290 7002; fax: +82 31 290 7015.

E-mail address: chkimbio@skku.edu (C.-H. Kim).

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doi:10.1016/j.phymed.2008.02.015
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Author:Kim, Kyung-Woon; Shin, Yong-Seung; Kim, Kap-Sung; Chang, Young-Chae; Park, Kwan-Kyu; Park, Jae-Bok;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:9SOUT
Date:Dec 1, 2008
Words:6554
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