7/27/2019 Egcg on Macrophages

http://slidepdf.com/reader/full/egcg-on-macrophages 1/4

Beneficial effects of green tea catechins on alveolarmacrophages infected with pneumonia causing bacteria.

Kazuto Matsunaga, ThomasW. Klein, Herman Friedman, and Yoshimasa Yamamoto

Department of Medical Microbiology and Immunology, University of South Florida College of

Medicine, Tampa, FL 33612, USA

Summary

Bacterial pneumonia in immunocompromised patients as well as elderly persons often

becomes a life threatening disease, even when effective antibiotics are used extensively. In

addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients

requires another approach to treat such patients besides treatment with antibiotics. In this

regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the

potential agents for such purpose due to its possible potential immunomodulatory as well as

antimicrobial activity. Current studies in this laboratory showed that EGCg enhanced the in

vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective

immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced

impairment of alveolar macrophages regarding antibacterial as well as immune activity was also

restored by EGCg treatment. These results indicate that EGCg may be a possible potential

immunotherapeutic agent against respiratory infections in immunocompromised patients, such

as heavy smokers. .

Keywords

Pneumonia, Legionellapneumophila, epigallocatechin gallate, a l v ~ o l a r macrophages, infection

Introduction

Alveolar macrophages are the preferential site for growth of pneumonia causingintracellular bacteria, such as Legionella pneumophila which is a causative agent of legionnaires

disease. It is widely accepted that the activation of macrophages is an essential effector

nlechanism for the resolution of infection with intracellular pathogens. Therefore, the

modulation of imnlune functions of macrophages may eventually affect the outcome of the

infection. Epigallocatechin gallate (EGCg) is a major form of green tea catechins and has a

variety of biological activities including antioxidant as well as anti-microbial activities against

some pathogens (3, 15). Although the biological activities of EGCg have extensively been

studied, its immunological effects are not well understood, particularly the beneficial effect on

immune cells regarding anti-microbial activity of cells. The studies conducted in this laboratory

revealed a possible iminunotherapeutic activity of EGCg on L. pneumophila infection of

alveolar macrophages (8). The results may introduce a possible new alternative

immunotherapeutic agent to the treatment of respiratory infections in patients who do not have a

strong immune defense against pathogens.

Materials and Methods

A mouse alveolar macrophage cell line MH-S was infected with L. pneumophila, as described

previously (7). The cells infected with bacteria were also treated with nicotine hydrogen tartrate

salt (Sigma, St. Louis, MO), cigarette smoking condensate ( C S C ~ Murty Pharmaceuticals,

Lexington, KY) and/or EGCg (Signla). In some experiments, macrophage cultures infected

- 1 5 1 - Session ill

7/27/2019 Egcg on Macrophages

http://slidepdf.com/reader/full/egcg-on-macrophages 2/4

with bacteria and treated with EGCg were incubated with anti-TNFa antibody. The cells

infected with bacteria and treated 'with CSC or nicotine were also incubated with mouse

recombinant TNFa (R & D Systems, Minneapolis, MN). The number of viable bacteria (CFU)

in the cultures as well as protein levels for cytokines in the culture supernatants were measured,

as described previously (9, 16).

Resultsand

DiscussionEGCg inhibits the growth of L. pneumophila. in alveolar macrophages. without any direct

effect on bacteria. L. pneumophila, a gran1-negative facultative intracellular pathogen, is the

causative agent of pneumonia in immunocompromised patients (2). The activation of

macrophages to suppress intracellular bacterial growth is an essential effector mechanism of the

resolution of legionellosis. The treatment of alveolar macrophage cell line (MH-S) cells with

EGCg (0.5 - 50 J!,g/ml) after infection with L. pneumophila induced a marked inhibition of the

growth of bacteria in the cells in a dose dependent manner. Even as little as a 0.5 JA,g/ml of

EGCg induced a significant inhibition of bacterial growth in macrophages. A study of a

possible direct anti-bacterial activity of EGCg on L. pneumophila showed that EGCg did not

possess any direct anti-L. pneumophila activity at up to the concentration of 500 JA,g/ml in

bacterial culture broth.

EGCg modulates production of certain cytokines induced by L. pneumophila infection. From

the previous .resuits, it seenled likely that EGCg might inhibit bacterial growth due to

macrophage activation. In order to determine such a possibility, the effect of EGCg on the

production of nlacrophage cytokines was examined. The treatment of macrophages with EGCg

alone slightly induced production of some cytokines at the high EGCg concentration, such as 50

J!,g/ml. However, EGCg markedly up-regulated the production of TNFa and IL-12 induced by

L. pneumophila infection, even at a concentration as little as 0.5 J.lg/ml in the case of TNFa. In

contrast, the IL-I0 production induced by bacterial infection was strongly down-regulated by

EGCg (0.5 - 50 J!,g/ml) in a dose dependent nlanner. On the other hand, the production of IL-6

induced by bacterial infection not affected by EGCg.Enhanced cytokine production is involved in the inhibition of bacterial growth by EGCg.

Since both endogenous and exogenous TNFa and IFNy are- known to activate macrophages to

inhibit L. pneumophila growth (4, 6, 9), involvement of enhanced TNFtx and IFNy in the

EGCg-induced bacterial growth inhibition was exanlined by neutralization with specific

antibodies to these cytokines. The treatment of nlacrophages with either anti-TNFa antibody or

anti-IFNy antibody almost completely abolished the EGCg (50 J!,g/ml)-induced bacterial growth

inhibition. These results clearly indicate that both TNFa and IFNy are involved in the anti-L.

pneumophila activity ofmacrophages induced by EGCg treatment.

EGCg treatment of macrophages infected with L. pneumophila enhances the level of IFNy

message. Since the treatment with anti-IFNy antibody induced a marked abolishment of the

EGCg induced-bacterial growth inhibition, levels of IFNy mRNA inmacrophages infected withbacteria and stimulated with or without EGCg were analyzed. The results showed that

messages for IFNy were induced in bacteria infected cells at 24 hrs after infection. Moreover,

when. the macrophages were infected and treated with EGCg, a remarkable enhancement of

IFNy mRNA accumulation was observed. However, EGCg (50 J!,g/ml) alone did not induce any

IFNy mRNA in macrophages.

EGCg restores the anti-L. pneumophila activity of alveolar macrophages suppressed by

treatment with nicotine or CSC. Tobacco smoking is known to cause deleterious effects on

immune cells followed by susceptibility to respiratory infections. For example, it is shown that

Session m - 1 5 2 -

7/27/2019 Egcg on Macrophages

http://slidepdf.com/reader/full/egcg-on-macrophages 3/4

snloking accelerates pneunlonia caused by S. pneumoniae (12). Pneumonia caused by other

bacteria, such as Chlanlydia, Legionella and Mycobacteria, is also known to frequently occur in

smokers (1, 5, 13, 14). However, the specific biological mechanisms by which exposure to

tobacco smoke increases the risk of bacterial pneumonia are poorly understood. In this regard,

our studies showed that both nicotine (1 J1g/ml) and CSC (2 J,lg/ml) induced a significant

increase of bacterial growth in alveolar macrophages associated with down-regulation of

production of certain cytokines. The EGCg treatment (0.5 - 50 J,lg/ml) restored and e v ~ nstrengthened the anti-L. pneumophila activity of macrophages suppressed by nicotine or CSC

treatment. Furthermore, EGCg (50 Jig/ml) diminished the nicotine or CSC-induced inhibition

of cytokine productions.

The results obtained in this study clearly showed that EGCg inhibited the growth of L.

pneumophila, which is an important pneumonia causing pathogen in immunocompromised

patients, in alveolar macrophages at a concentration as low as 0.5 Jig/nll, which can be reached

in plasma by ingestion of capsules of tea extracts (10, 11, 17, 18). The EGCg s e l e ~ t i v e l y up

regulated the production of IL-12 and TNFa and down-regulated IL-I0 production of

macrophages induced by bacterial infection, but did not alter IL-6 production. The up

regulation of the message levels of IFNy by EGCg was also demonstrated. Treatment of

macrophages with anti-TNFa and anti-IFNy antibodies abolished the anti-bacterial activity of

macrophages induced by EGCg treatment. These results indicate that EGCg selectively alters

the immune responses of macrophages 10 pathogens and leads to enhanced anti-bacterial

activity of macrophages mediated by enhanced production of both TNFa and IFNy.

Furthermore, EGCg treatment restores the anti-bacterial activity of macrophages suppressed by

nicotine or CSC treatment, which is a mimic of alveolar cells in tobacco smokers and reversed

by EGCg. Thus, these results revealed the selective immunomodulatory effect of EGCg on

alveolar macrophages, which have a critical role in respiratory infections. The study warrants a

further study of EGCg as a potential alternative imnlunotherapeutic agent in respiratory

infections.

Acknowledgement

This work was supported by grants AI45169 from the National Institute of Allergy and

Infectious Diseases and the American Lung A s s o c i a t i ~ n of Florida.

References

1. Altet, M. N., J. Alcaide, P. Plans, J. L. Taberner, E. SaIto, L. I. Folguera, and L. Salleras.

1996. Passive smoking and risk of pulmonary tuberculosis in children immediately

following infection. A case-control study. Tuber Lung Dis. 77:537-44.

2. Friedman, H., Y. Yamamoto, C. Newton, and T. Klein. 1998. Immunologic response

and pathophysiology ofLegionella infection. Semin Respir Infect. 13: 100-8.

3. Hamilton-Miller, J. M. 1995. Antimicrobial properties of tea (Camellia sinensis L.).

Antimicrob Agents Chemother. 39:2375-7.

4. Klein, T. W., Y. Yamamoto, H. K. Brown, and H. Friedman. 1991. Interferon-gamma

induced resistance to Legionellapneumophila in susceptible NJ mouse macrophages. J

Leukoc BioI. 49:98-103.

5. Leinonen, M., and P. Saikku. 1999. Interaction of Chlamydia pneumoniae infection

with other risk factors of atherosclerosis. Am Heart J. 138:S504-6.

- 1 5 3 - Session il l

7/27/2019 Egcg on Macrophages

http://slidepdf.com/reader/full/egcg-on-macrophages 4/4

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Session ill

Matsiota-Bernard, P., C. Lefebre, M. Sedqui, P. Cornillet, and M. Guenounou. 1993.

Involvement of tumor necrosis factor alpha in intracellular multiplication of Legionella

pneumophila in human monocytes. Infect Immun. 61:4980-3.

Matsunaga, K., T. W. Klein, H. Friedman, and Y. Yamamoto. 2001. Alveolar

macrophage cell Line MH-S is valuable as an in vitro model for Legionella

pneumophila infection. Am J Respir Cell Mol BioI. 24:326-31.

Matsunaga, K., T. W. Klein, H. Friedman, and Y. Yamamoto. 2001. Legionellapneumophila replication in macrophages inhibited by selective immunomodulatory

effects on cytokine formation by epigallocatechin gallate, a major fonn of tea catechins.

Infect Immun. 69:3947-53.

McHugh, S. L., C. A. Newton, Y. Yanlamoto, T. W. Klein, and H. Friedman. 2000.

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila

infection. Proc Soc Exp BioI Med. 224: 191-6.

Nakagawa, K., and T. Miyazawa. 1997. Absorption and distribution of tea catechin, (-)epigallocatechin-3- gallate, in the rat. J Nutr Sci Vitaminol (Tokyo). 43:679-84.

Nakagawa, K., S. Okuda, and T. Miyazawa. 1997. Dose-dependent incorporation of tea

catechins, (-)-epigallocatechin-3- gallate and (-)-epigallocatechin, into human plasma.

Biosci Biotechnol Biochem. 61: 1981-5.

Nuorti, J. P., J. C. Butler, M. M. Farley, L. H. Harrison, A. McGeer, M. S. Kolczak, and

R. F. Breiman. 2000. Cigarette smoking and invasive pneumococcal disease. Active

Bacterial Core Surveillance. TeamN Engl J Med. 342:681-9.

Pedro-Botet, M. L., M. Sabria-Leal, M. Haro, C. Rubio, G. Gimenez, N. Sopena, and J.

Tor. 1995. Nosocomial and community-acquired Legionella pneumonia: clinical

comparative analysis. Eur Respir J. 8: 1929-33.

Straus, W. L., J. F. Plouffe, T. M. File, Jr., H. B. Lipman, B. H. Hackman, S. J.

Salstrom, R. F. Benson, and R. F. Breiman. 1996. Risk factors for domestic acquisition

of legionnaires disease. Ohio legionnaires Disease Group. Arch Intern Med. 156: 1685

92.Weisburger, J. H. 1999. Second international scientific symposium on tea and human

health: An introduction. Proc Soc Exp BioI Med. 220: 193'-4.Yamanl0to, Y., T. W. Klein, C. A. Newton, R. Widen, and H. Friedman. 1987.

Differential growth of Legionella pneunl0phila in guinea pig versus mouse nlacrophage

cultures. Infect Immun. 55:1369-74.

Yang, C. S., L. Chen, M. J. Lee, D. Balentine, M. C. Kuo, and S. P. Schantz. 1998.

Blood and urine levels of tea catechins after ingestion of different amounts of green tea

by human volunteers. Cancer Epidemiol Biomarkers Prevo 7:351-4.

Zhu, M., Y. Chen, and R. C. Li. 2000. Oral absorption and bioavailability of tea

catechins. Planta Med. 66:444-7.

- 1 5 4 -