Gerimax Ginseng Regulates Both Humoral and Cellular Immunity ...

THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 8, Number 4, 2002, pp. 459–466 © Mary Ann Liebert, Inc.

Gerimax Ginseng Regulates Both Humoral and Cellular Immunity During Chronic Pseudomonas aeruginosa Lung Infection ZHIJUN SONG, M.D., Ph.D.,1,2 HONG WU, M.D.,2 KALAI MATHEE, Ph.D.,1,2 NIELS HØIBY, M.D., Dr.Med.Sci.,2 and ARSALAN KHARAZMI, Ph.D.2

ABSTRACT Background: Chronic lung infection among patients with cystic fibrosis (CF), diffused panbronchiolitis, and chronic obstructive bronchiecteisis is often because of Pseudomonas aeruginosa. High morbidity and mortality in patients with CF are because of P. aeuruginosa that undergoes genotypic and phenotypic changes during prolonged stay in the lung resulting in increased antibiotic resistance, necessitating a search for alternative or supplement drugs. Objective: In this study we compared the therapeutical effect of Gerimax (Dansk Droge A/S, Ishøj, Denmark) ginseng with placebo control by using a rat model of chronic P. aeruginosa lung infection mimicking that in patients with CF. Methods and interventions: The animals were challenged intratracheally with the prototypic P. aeruginosa PAO1 in alginate beads (1 3 109 colony-forming units per milliliter [CFU/mL]) followed by subcutaneous injection of ginseng extract (150 mg/kg body weight once per day) and examined on days 7 and 21. Results: The day 7 analyses show that ginseng treatment resulted in lowering serum immunoglobulin M (IgM) and lung interleukin-4 (IL-4) levels compared to the control group. On day 21, higher lung IgA, upregulated serum IgG2a, stronger lung responses of interferon-g, IL4, and tumor necrosis factor-a with milder lung pathology and enhanced lung bacteriology were detected in the ginseng-treated group when compared to those of the control group. Conclusion: These results suggest that the Gerimax ginseng treatment can modulate the immune system in favor of clearing the infection with P. aeruginosa in the lungs of rats. Thus, ginseng might be a promising alternative supplement for the treatment of chronic P. aeruginosa lung infection in patients with CF.

INTRODUCTION

P

seudomonas aeruginosa is ubiquitous in nature and an important opportunistic pathogen that causes urinary-tract, respiratorysystem, dermatitis, and soft-tissue infections. It is also responsible for a variety of systemic in1 Department 2 Department

fections particularly in victims of severe burn, and in cancer and patients with acquired immune deficiency syndrome (AIDS) who are immunosuppressed. Chronic lung infection with P. aeruginosa is common among patients with cystic fibrosis (CF), diffused panbronchiolitis, and chronic obstructive bronchiecteisis. P.

of Biological Sciences, Florida International University, Miami, FL. of Clinical Microbiology, University Hospital (Rigshospitalet), Copenhagen, Denmark.

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aeruginosa is the leading pathogen responsible for morbidity and mortality among patients with CF. Although CF occurs in all ethnic groups and geographic locations, its highest incidence is among Caucasians as the most common fatal inherited disease, afflicting 1 in 2000 and 1 in 3700 live births in the United States and Denmark, respectively. CF is associated with a defect in a cyclic adenosine monophosphate (cAMP)-activated chloride channel in secretory epithelia and this defect leads to decreased fluid secretion; the dehydration of epithelial surfaces initiates the pathology of the disease (Frizzell, 1999). Oversecretion of mucus in the airway leads to congestion of the respiratory tract and increased susceptibility to bronchopulmonary infection, which is the major cause of morbidity and mortality among patients with CF. Bacteria commonly isolated from sputum of patients with CF are Staphylococcus aureus, Haemophilus influenzae, and P. aeruginosa (Koch and Høiby, 1993). Both S. aureus and H. influenzae can be effectively eradicated by using oral antibiotics. However, P. aeruginosa infection, which occurs in 60% to 90% of patients with CF, is never eradicated despite intensive antipseudomonal treatment (Høiby, 1993; Koch and Høiby, 1993). Immune responses can be divided into T helper cell type TH 1 and TH 2 types. It has been known that the immune response against chronic P. aeruginosa lung infection in patients with CF is a TH 2-like response (Moser et al., 2000). The TH 1 type is characterized by the production of antigen-specific IgG2a and the secretion of interferon-g (IFN-g), interleukin (IL)12, and IL-2, which favor cellular immunity, whereas the TH 2 type is associated with antigen-specific IgG1 production, the secretion of IL-4 and IL-10 as well as the proliferation of B cells and mast cells, which favor humoral immunity (Belardelli, 1995). In the hope of finding new and efficacious measures for the treatment of P. aeruginosa pneumonia in patients with CF, scientists have explored alternative treatments. It has been found that the immunization with P. aeruginosa vaccines and adjuvants, or the treatment with IFN-g or the Chinese herbal medicine, Daphne giraldii Nitsche, could decrease the inflammatory response and enhance the bacterial clearance in a rat model

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(Johansen et al., 1994, 1995, 1996; Song et al., 1996). Treatment with IFN-g, Radix anglicae Sinensis, Shuanghuanglian, and crude ginseng extract can change the TH 2 response to a TH 1 type and therefore change to a more favorable outcome during chronic P. aeruginosa lung infection (Johansen et al., 1996; Song et al., 1997, 2000). Ginseng is one of the best-known Chinese medicinal herbs and it has been widely used in China for thousands of years (Huang, 1993; O’Hara et al., 1998). The toxicity of ginseng is low, the median lethal dose (LD50) for intravenous injection in mice is 16.5 mg/kg (Bensky and Gamble, 1993). The herb is reported to influence the cardiovascular, nervous, endocrine, and the immune systems. In addition, it affects metabolism, possesses some anticancer, antiinflammation, and antiaging effects (Huang, 1993). As a modulator of the endocrine system and immune system, ginseng has been shown to have some functions in modulating the production of specific antibodies and the functions of phagocytes and natural killer (NK) cells (Huang, 1993; Yang et al., 1983, 1987). It appears that an aqueous extract of ginseng (Panax ginseng [C.A. Meyer]) is able to suppress overactive immune response in a chronic infection rat model (Song et al., 1997). The ginsengtreated animals had lower total serum IgG and higher IgG2a, decreased the mast cell number in the lung foci, and induced a change in the pulmonary inflammation with a shift from a polymorphonuclear to a mononuclear leukocyte infiltration. This is in accordance with a change from a TH 2-like to a T H 1-like response. Because production of IgG2a is associated with IFN-g, the authors postulated that ginseng mimics IFN-g, a TH 1 immune response indicator. Thus, it is possible for ginseng to induce a TH 1 response in patients with CF. However, the cytokines, which are the actual indicators of immune response were not measured. Ginseng, imported or cultivated and produced en masse, is now available in many countries. The ginseng used in our previous studies came from the Changbai Mountains in Jilin Province of China. Unfortunately, we were not able to get any information on the preparation except for assurance of the quality of the ginseng supplied. In order to standardize all

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GINSENG AND CHRONIC P. AERUGINOSA LUNG INFECTION

future work, we need ginseng extract of the same quality from batch to batch. We decided to investigate the efficacy of Gerimax ginseng marketed by Dansk Droge A/S, Ishøj, Denmark. It is from Panax ginseng C.A. Meyer (Araliaceae) and each Gerimax ginseng preparation is standardized to contain 8.5% (w/w) ginsenosides. In the present study, the effect of a Gerimax ginseng extract on the rat model of P. aeruginosa (PAO1) lung infection was investigated. Our studies indicate that this ginseng preparation modulates both cellular and humoral immune responses.

MATERIALS AND METHODS Bacterial strain The prototypic, nonmucoid P. aeruginosa PAO1 (PAO1) (Holloway and Matsumoto, 1984) was used. Animals and experimental groups Female Lewis rats (Charles River, Würtzburg, Germany), 7 weeks old with a body weight of approximately 150 g, were used in these investigations. The number of animals and the experimental protocols are given in Table 1.

TABLE 1. PROTOCOL USED Experiment I

II

Immobilization of bacteria in seaweed alginate Immobilized P. aeruginosa in seaweed alginate beads was prepared as previously described (Pedersen et al., 1990). Briefly, overnight bacterial culture grown in Luria broth was harvested, resuspended in fresh medium, and 1 mL of the cells was mixed with 9 mL of sterile seaweed alginate (Protanal 10/60; Protan, Drammen, Norway). The mixture was forced with air through a cannula/ channel into a solution of 0.1 mol/L CaCl2 in 0.1 mol/L Tris-HCl buffer (pH 7.0). The suspension was adjusted to yield 109 colony-forming units per milliliter (CFU/mL). Challenge procedures Before challenge, all rats were anesthetized subcutaneously with a 1:1 mixture of etomidate and midazolam at a dose of 1.5 mL/kg body weight, and tracheotomies were performed (Johansen et al., 1993). Intratracheal challenge with 0.1 mL of P. aeruginosa (109 CFU/mL) in alginate beads was performed as previously described (Johansen et al., 1993). The incisions were sutured with silk and healed without any complications. All rats were killed on different days by administering 20% (v/v) pentobarbital (DAK, Copenhagen, Denmark) at 3 mL/kg body weight and blood samples were obtained by cardiac puncture.

IN THE

ANIMAL EXPERIMEN T

Bacteriala strain

No. of animalsb

Group

Comment

PAO1

10

Ginseng c

The animals were killed 7 days postchallenge to evaluate the effect of early infection

PAO1

10 10

Salined Ginseng

10

Saline

The animals were killed 21 days postchallenge to evaluate the effect of chronic infection

a Pseudomonas aeruginosa was immobilized in seaweed alginate beads and rats were challenged intratracheally as described in Materials and Methods. b The animals used were female Lewis rats (Charles River, Würtzburg, Germany), 7 weeks old with a body weight of approximately 150 g. c Gerimax ginseng powder containing 8.5% (w/w) of ginsenosides was provided by Dansk Droge A/S, Ishøj, Denmark. Ginseng extract at 150 mg/kg body weight was injected subcutaneously once per day as described in Materials and Methods. d The saline for the control group was injected subcutaneously at 1 mL/kg body weight once per day. The injections were given on the same day when all animals were challenged with P. aeruginosa alginate beads.

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Gerimax ginseng extract The ginseng powder used was from Panax ginseng C.A. Meyer from Araliaceae provided by Dansk Droge A/S. The main and side roots were extracted with 40% ethanol. This extract contains ginsenosides and other root components such as polysaccharides, sterols, organic acids, phenolic acids, flavonoids, essential oils, vitamins, and trace elements. Each preparation was standardized such that it contained 8.5% (w/w) ginsenosides based on the high-pressure liquid chromatograpy (HPLC) analysis. The extract was then dried and ground into a powder in the presence of 40%–50% (w/w) maltodextrin as a technical carrier component. This preparation was completely soluble in water. The ginseng extract was dissolved in saline to give a stock solution of 50 mg/mL. The ginseng extract was injected subcutaneously at 150 mg/kg body weight once per day. The saline or the control group was injected subcutaneously at 1 mL/kg body weight once per day. The injections were started on the same day when all animals were challenged with P. aeruginosa alginate beads. Macroscopic lung pathology The macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP), which was calculated according to the following formula: LIMP 5 the lung area with pathologic changes/the area of the same side lung (left lung) as previously described (Grimwood et al., 1989; Song et al., 1998). In addition, the gross pathologic changes in the lungs were also divided into four different groups according to the severity of inflammation as previously described (Johansen et al., 1994; Song et al., 1997): 1, normal; 2, swollen lungs, hyperemia, small atelectasis (,10 mm2); 3, pleural adhesions and atelectasis (,40 mm2); 4, abscesses, large atelectasis and hemorrhages. Lung bacteriology Lung samples from each group were prepared for quantitative bacteriologic examination as previously described (Johansen et al., 1994). Lungs were homogenized in 5 mL of phosphate-buffered saline, and appropriately diluted samples were plated in duplicate on

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Blue agar plates (a modified Conradi-Drigalski medium selective for gram-negative rods containing lactose, pH 7.0; State Serum Institute, Copenhagen, Denmark) to determine the number of CFU after 20–24 hours of incubation at 37°C. Our detection limit was 100 CFU/mL or more of lung homogenate (Johansen et al., 1994; Song et al., 1997). The remainder of lung homogenate was then centrifuged and the supernatant was kept for cytokine and antibody examination. Antibody responses Quantitation of anti-P. aeruginosa antibodies, Immunoglobulin M (IgM), IgG and IgA classes, and IgG1, IgG2a subclasses in the serum was carried out by means of enzyme-linked immunosorbent assay (ELISA) as previously reported (Johansen et al., 1994; Song et al., 1997). The P. aeruginosa PAO1 antigens were prepared by using a 100-mL overnight culture that was washed with saline and resuspended in 5 mL of saline, and sonicated to release all the proteins. The sonicate was centrifuged to remove the particulate matter and the supernatant was used as antigens for ELISA. The antibody concentrations expressed as ELISA units (EU) were obtained by dividing the mean optical density of the samples with the mean optical density of an internal standard expressing absorbance units between 0.30–0.40. Cytokine responses The concentrations of cytokines, IFN-g, IL-4, and TNF-a, in the lung homogenate were determined by ELISA kits (Nordic BioSite, Sweden). Standard curves for IL-4 ranging from 8–500 pg/mL (lower detection limit, 2 pg/mL), IFN-g ranging from 10–2000 pg/mL (lower detection limit, 10 pg/mL), and tumor necrosis factor-a (TNF-a) ranging from 20–1000 pg/mL (lower detection limit, 20 pg/mL), were constructed. The optical density (OD) value of each sample was compared with the standard curve of the same cytokine in the same ELISA plate to obtain the cytokine unit (pg/mL). Statistical analyses Unpaired differences in continuous data were analyzed by the Mann-Whitney U test

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and categorical data were compared using the x2 test. The chemotaxis data were compared using a paired two-tail Student’s t test. p # 0.05 was considered significant.

ginseng-treated group (50%) compared to the control group (82%). However on day 21, significantly milder lung pathology (lower LIMP value) was found in the ginseng-treated compared to the control group (p , 0.03) (Table 2). Lung bacteriology

RESULTS Mortality None of the animals in the ginseng-treated group died from lung infection but one in the untreated control group died. The low mortality rate suggests that the amount of bacteria used was sufficient to create the infection but not enough to kill the animals. Macroscopic lung pathology The major pathologic changes observed were lung consolidation, abscesses, adhesion, hemorrhage, and atelectasis. The severity of the lung pathology was scored as the LIMP index (described in Materials and Methods). On day 7, lung consolidation, hemorrhage, and edema were the main pathologic change and there was no significant difference between the ginsengtreated and control groups (Table 2). On day 21, although the lung abscesses were not significantly different, the incidence was lower in TABLE 2. PARAMETERS MEASURED

ON

DAYS 7

AND

We enumerated the total bacteria in lung tissues to evaluate the ability of ginseng to enhance the immune response in bacterial clearance. On day 7, the lung bacteriology did not differ between the two groups with and without ginseng treatment (Table 2). However, on day 21 the lung bacterial clearance was enhanced greatly by the ginseng treatment (p , 0.035) when compared with the nontreated control group (Table 2). Specific antibody responses Serum antibody levels against total PAO1 sonicate were measured 7 (only IgM) and 21 (IgA and IgGs) days postinfection. On day 7, serum IgM levels, indicator of early immune response, in the ginseng-treated group was significantly downregulated (p , 0.02) compared to the control group (Table 2). On day 21, higher IgA level was found in the supernatant of lung homogenate from the ginseng-treated 21 POSTCHALLENGE

P.

AERUGINOSA

PAO1

Parametera

Day

LIMP

7 21 7 21

0.41 0.07 6.3 1

(0.18–0.49) (0.05–0.1) 3 105 (4–170 3 104) 3 103 (0–2.5 3 105)

0.31 0.09 7.2 4.6

(0.14–0.41) (0.06–0.22) 3 105 (8.7–90 3 104) 3 104 (5.8–13,000 3 102)

. 0.06 0.029 . 0.9 0.0346

7 21 21 21 21

35 35 1368 59 111

(17–127) (6–45) (652–2010) (17–115) (78–157)

56 16 1372 46 97

(43–219) (1–39) (749–2249) (9–156) (64–124)

, 0.02 0.05 . 0.6 . 0.8 0.05

7 21 7 21 7 21

859 1196 153 268 215 338

(705–2355) (876–3596) (97–224) (174–491) (27–423) (235–741)

989 621 215 137 261 207

(709–1262) (246–1109) (137–304) (108–210) (167–304) (170–517)

. , , , . ,

Bacteriology Serum antibody levels: IgM IgA IgG IgG1 IgG2a Cytokine levels: IFN-g IL-4 TNF-a a The

Ginsengb

WITH

Salineb

p valuec

0.8 0.002 0.05 0.0004 0.3 0.004

various parameters were measured and expressed as described in Materials and Methods. results are expressed as the median with a range given in parentheses. c The p values were calculated using Mann-Whitney U test. LIMP, lung index of macroscopic pathology; Ig, immunoglobulin; IFN-g, interferon-g; IL-4, interleukin-4; TNF-a, tumor necrosis factor-a. b The

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SONG ET AL.

group (p 5 0.05) compared to the control (Table 2). There was no obvious difference between the two groups in serum IgG and IgG1 on day 21; however, the IgG2a level in the ginsengtreated group was higher than that in the control group (p 5 0.05) (Table 2). Cytokine responses Cytokines are the indicators of the type of immune response elicited during infection. On day 7 PAO1 postinfection, ginseng treatment reduced the production of IL-4, indicator of TH 2like immune response, in the lung tissues compared to the control group (p , 0.05) (Table 2). There was no significant difference between the two groups in lung IFN-g and TNF-a responses. On day 21, however, ginseng treatment enhanced the production of all three cytokines in the lung tissues significantly (Table 2).

DISCUSSION Chronic P. aeruginosa lung infection is the major cause of morbidity and mortality in patients with CF. The infection results in inflammation in the lungs characterized by the infiltration of polymorphonuclear leukocytes (PMNs) in the lung tissues and a significant antibody response against P. aeruginosa (Høiby, 1995; Pedersen, 1992), leading to immune complex formation and lung tissue damage. In addition, the outcome of immune response is characterized by the cytokine secretion pattern of the TH -cell subsets. In a recent paper, Mullen et al. (2001) argued that cytokines determine not only the fate of TH cells but also mediate selective survival of a lineage. The TH 2 type is associated with antigen-specific IgG1, IgM, IgA, and IgE production, the secretion of IL-4 and IL-10 that favor humoral immunity whereas TH 1 type is characterized by the production of antigen-specific IgG2a and the secretion of IFN-g, IL-12, and IL-2, which favor cellular immunity (Belardelli, 1995; Goldsby et al., 2000). IL-4 and IFN-g, are essential for the differentiation of TH 2 and TH 1, respectively. It has been known that the immune response against chronic P. aeruginosa lung infection in patients with CF is a TH 2-like response (Moser

et al., 2000). Using animal models we have shown that the immune response can be redirected to a more favorable outcome, from the TH 2 to TH 1 type, during chronic P. aeruginosa lung infection by treatment with IFN-g, Radix anglicae Sinensis, Shuanghuanglian, and crude ginseng extract (Song et al., 1997, 2000). The earlier ginseng work was done with Panax ginseng (C.A. Meyer) powder provided by Millingwang Limited, Jilin, People’s Republic of China and P. aeruginosa PAO579. This work was initiated to determine if the ginseng extract from an alternate provider would have a similar outcome with another P. aeruginosa isolate. Our choice of the strain was influenced by the fact that P. aeruginosa PAO1 is the prototypic strain whose genome was recently published. The current work was repeated using Panax ginseng (C.A. Meyer), provided by Dansk Droge A/S. This preparation is sold over the counter and is popularly known as Danish Gerimax ginseng. Each Gerimax ginseng preparation is standardized to 8.5% (w/w) of ginsenosides based on the HPLC analysis. It has been reported that treatment with Gerimax ginseng could improve the reaction of central nervous system (Sandberg and Dencker, 1994; Sørensen and Sonne, 1996) and that this preparation has shown therapeutic effect on patients with noninsulin–dependent diabetes (Sotaniemi et al., 1995). This study was carried out to look at the effect of Gerimax ginseng on the 7th and 21st day postinfection. This is the first study to show the effect of this ginseng product on the outcome of humoral and cellular immunity during chronic bacterial infection. Ginseng treatment reduces lung pathology and enhances bacterial clearance We looked at the outcome of ginseng treatment by using a well-established chronic animal infection model for CF. In this model, the lungs of the animals are instilled with P. aeruginosa coated with alginate beads, which give rise to immune response similar to that seen in patients with CF (Johansen et al., 1993). However, alginate beads alone without bacteria do not lead to any pathologic changes (data not shown). Our study shows that the ginseng treatment resulted in significantly milder lung pathology. In addi-

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tion, this study also shows enhanced bacterial clearance on day 21. Similar results were obtained in our previous study with the same animal model but with PAO579 and aqueous extract of Panax ginseng that the ginseng-treated animals exhibited a significantly enhanced bacterial clearance, less severe lung pathology, lower lung abscess incidence compared to the control group of animals (Song et al., 1997). The effect on the bacterial persistence in lungs during treatment suggests that ginseng may be involved in downregulating expression of bacterial genes necessary for prolonged survival. Future studies will focus on the role of ginseng in modulating virulence genes, in vivo and in vitro. Ginseng modulates humoral immune response during chronic bacterial infection In order to correlate the pathologic changes with primary and secondary humoral immune responses, we looked at the serum IgM, IgA, and IgG levels against P. aeruginosa antigens (Table 2). As predicted from the bacterial and pathologic analyses, the treatment with this ginseng preparation on the animals chronically infected with P. aeruginosa PAO1 resulted in modulating humoral immune response. The downregulation of serum IgM on day 7 and upregulation of serum IgG2a and lung IgA levels on day 21 suggest that ginseng may also play a role in the suppression of overreactive humoral immune response. Similar results were obtained in our previous study with Panax ginseng (C.A. Meyer); the ginseng-treated animals had lower total serum IgG and higher IgG2a. In both these studies, it appears that ginseng is able to modulate the immune response, but the outcome is largely dictated by the choice of ginseng. Ginseng modulates cellular immune response during chronic bacterial infection Because production of IgG2a is associated with IFN-g, it is postulated that ginseng mimics IFN-g, a TH 1 immune response indicator. In order to correlate the pathologic changes and humoral with cellular immune responses, we looked at the cytokine levels. The lung IL-4 levels on day 7 were significantly lower in the ginseng treated animals that were chronically in-

fected with P. aeruginosa PAO1. The lower IL4 levels is in accordance with the shift from TH 2 to TH 1. However, we did not see a concomitant rise in the IFN-g. Interestingly, IFN-g, IL-4, and TNF-a in the lung tissues on day 21 increased in comparison with the control group suggesting that both TH 1 and TH 2 responses were activated significantly. Perhaps, the synergistic effect of both systems by ginseng may provide a protective effect. In the present study, the downregulation of serum IgM on day 7 after PAO1 infection is associated with the reduction of IL-4 in the lungs suggesting an early onset of the favorable TH 1 response. The increased production of IFN-g, IL4, and TNF-a in the lung tissues on day 21 suggests an activation in both cellular and humoral immunity. IgA is a local antibody on mucous membrane and it is an important part of local immunity of airway. The increase in lung IgA might be helpful for the animal to neutralize and clear the bacteria from the lungs. IgG1 and IgG2a are involved in TH 2 and TH1 responses, respectively. The increase of serum IgG2a or decrease of IgG1 against P. aeruginosa indicates a systemic TH 1-like response. The active immune system as seen 21 days postinfection in the ginseng-treated animals might be responsible partly for the milder lung pathology and enhanced bacterial clearance from the lungs. Our results suggest that ginseng might be a promising alternative supplement for the treatment of chronic P. aeruginosa lung infection in patients with CF. It will be interesting to analyze the effect of ginseng during infection with antibiotic resistant P. aeruginosa strains in the presence and absence of antibiotics. ACKNOWLEDGMENTS We thank Jette Pedersen and Anne Asanovski for their expert technical assistance, Dr. Chong-Lek Koh for critical reading of the manuscript, and Dansk Dorge A/S, Ishøj, for providing ginseng. REFERENCES Belardelli F. Role of interferons and other cytokines in the regulation of the immune response. APMIS 1995;103: 161–179.

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Address reprint requests to: ZhiJun Song, M.D., Ph.D. Department of Biological Sciences, OE118 College of Arts and Sciences Florida International University University Park Miami, FL 33199 E-mail: [email protected]