Beneficial effects of green tea catechins on alveolar

macrophages infected with pneumonia causing bacteria.

Kazuto Matsunaga, Thomas W. 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, Legionella pneumophila, epigallocatechin gallate, macrophages, infection
Introduction
Alveolar macrophages are the preferential site for growth of pneumonia causing
intracellular 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 Murty Pharmaceuticals,
Lexington, KY) and/or EGCg (Signla). In some experiments, macrophage cultures infected
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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).
Results and Discussion
EGCg 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 w a ~ 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 of macrophages 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 with
bacteria 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
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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
strengthened 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 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 of Florida.
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