Nitric oxide synthase and cytokines gene expression analyses in Leishmania-infected RAW 264.7 cells treated with an extract of Pelargonium sidoides (Eps[R] 7630).
A modern aqueous-ethanolic formulation of the roots of Pelargonium sidoides (Eps[R] 7630), elaborated from the traditional herbal medicine used in areas of southern Africa, is effectively employed for the treatment of ENT and respiratory tract infections in modern phytotherapy. Previous studies have demonstrated antibacterial and immunomodulatory activities. To gain insight into the mode of action at the molecular level, gene expression analyses for the inducible nitric oxide synthase and the cytokines interleukin (IL)-1, IL-12, IL-18, tumour necrosis factor (TNF)-[.sub.-[alpha]], interferon (IFN)-[.sub.-[alpha]], and IFN-[.sub.-[gamma]], were performed using reverse transcription-polymerase chain reaction (RT-PCR). The experiments were carried out in parallel in non-infected and in Leishmania major-infected RAW 264.7 cells and the expression profiles were compared with those mediated by IFN-[.sub.-[gamma]] + LPS. Eps[R] 7630 induced low mRNA levels in non-infected cells, and it considerably up-regulated the transcript expressions in parasitised cells. Interestingly, and in contrast to activation by IFN-[.sub.-[gamma]] + LPS, Eps[R] 7630 also stimulated infected cells to produce IFN-[.sub.-[gamma]] mRNA. A similar expression profile was observed for the methanol-insoluble fraction (MIF) of Eps[R] 7630 and gallic acid, a trace constituent of the extract, while the methanol-soluble fraction and umckalin, an exclusive and representative member of the occurring coumarins, proved to be devoid of any remarkable gene-inducing capabilities. The present results provide not only convincing support for the improvement of immune functions as previously demonstrated in functional bioassays, but also evidence for activation at the transcriptional level and suggest that the underlying inducing principle is located in the MIF.
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Keywords: Pelargonium sidoides; Immunomodulation; Gene expression analysis; Nitric oxide synthase; Cytokines
Pelargonium sidoides DC (Geraniaceae) is a herbaceous commonly black-flowered perennial with a tuberous rootstock. Root decoctions of this species have a long tradition of use in the treatment of gastrointestinal disorders, chest pain, bronchial infection, and related troublesome respiratory tract conditions among several ethnic groups in areas of southern Africa, including Zulu, Bantu, Xhosa and Mfengu (Watt and Breyer-Brandwyk, 1962; Hutchings, 1996; Kolodziej and Kayser, 1998; Kolodziej, 2002). The therapeutic importance is shown by the introduction of an extract of the roots of P. sidoides, termed Eps[R] 7630 (Trademark: Umckaloabo[R], Iso-Arzneimittel, Ettlingen, Germany), into modern phytotherapy. Eps[R] 7630 is successfully employed in Germany, in the Commonwealth of Independent States, in Baltic states, and in Mexico for the treatment of ENT and respiratory tract infections. Numerous clinical studies have provided convincing evidence of the beneficial effects of Eps[R] 7630 therapy in patients afflicted with chronic tonsillitis, bronchitis, sinusitis, and rhinopharyngitis (Heil and Reitermann, 1994; Dome and Schuster, 1996; Haidvogl et al., 1996; Blochin et al., 1999; Blochin and Heger, 2000; Heger and Bereznoy, 2002; Golovatiouk and Chuchalin, 2002; Matthys et al., 2003; Kolodziej and Schulz, 2003). However, the mode of action of Eps[R] 7630 is only partially understood. Pharmacological studies have demonstrated antibacterial and immune modulatory activities for P. sidoides extracts and some representative constituents (Kayser and Kolodziej, 1997; Kolodziej and Kayser, 1998; Kayser et al., 2001; Kolodziej et al., 2003).
In this paper, we report on gene expressions of transcripts of iNOS and a series of cytokines induced by Eps[R] 7630, providing insight into the underlying molecular mechanisms of the demonstrated immune modulatory activities.
Materials and methods
Extracts, test compounds and chemicals
Eps[R] 7630, a special aqueous-ethanolic (11% m/m) extract of P. sidoides roots with a yield of 9-11%, was kindly provided by ISO Arzneimittel, Ettlingen, Germany. The extract (200 g) was partially dissolved in methanol (MeOH) (400 ml) and the suspension stirred overnight at room temperatures to afford a MeOH-soluble (MSF) (ca. 70 g) and a MeOH-insoluble fraction (MIF) (ca. 130 g). Stock solutions (20 mg/ml) in DMSO were prepared and kept at 4 [degrees]C until use. Gallic acid and umckalin were obtained from the parent extract, identified by analysis of their spectroscopic data and comparison with those of authentic specimens (Kayser and Kolodziej, 1995). All samples were first subjected to assays for endotoxin contamination (Limulus amoebocyte lysate method), which may stimulate immune cells, of which we found no evidence. Recombinant murine interferon (IFN)-[.sub.-[gamma]] (Genentech, San Francisco, USA), expressed in Escherichia coli, was kindly provided by Bender & Co., Wien, Austria. Trizol RNA isolation reagent was obtained from Invitrogen (Karlsruhe, Germany), reverse transcriptase (murine lymphoma virus RT RNAseH-), random hexamer primers and Taq polymerase, expressed in E. coli, and reverse transcription (RT) and polymerase chain reaction (PCR) buffers were supplied by Promega, Madison, USA. The oligonucleotides used as primers were synthesised by TiBMolbiol, Berlin, Germany.
General experimental procedures including cell cultures, parasites and in vitro infection of macrophages with Leishmania major parasites are described in details (Kolodziej et al., 2001).
Incubation of Leishmania-infected RAW 264.7 cells with samples
Infected RAW 264.7 cells were incubated in medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum) alone, or with medium containing 50 [micro]g/ml of the samples or with IFN-[.sub.-[gamma]] (100 U/ml) plus LPS (10 ng/ml) as positive control for 48 h at 37 [degrees]C. In parallel cultures, non-infected cells were similarly treated with or without samples as well as the control stimuli. Total RNA was extracted from independent cultures after 4h of exposure. Incubations were stopped by discarding the culture supernatant and freezing the cell monolayer until analysis.
RNA isolation and cDNA synthesis
Total RNA was extracted from RAW 264.7 cell preparations using an RNA purification kit (Invitrogen, Karlsruhe, Germany). In brief, 5 x [10.sup.5] cells were lysed in 1 ml of Trizol, followed by addition of chloroform (200 [micro]l), and centrifugation (12000g; 4 [degrees]C) of the suspension for 15min. The upper hydrophilic layer was recovered, mixed with isopropyl alcohol (500 [micro]l), again centrifuged, and the supernatant discarded. The pellet was washed twice with 75% ethanol, dissolved in [H.sub.2]O, adjusted to 1 mg RNA/ml and stored at -80 [degrees]C. RT was performed at 37 [degrees]C for 1 h in a total volume of 25 [micro]l in RT buffer [7mM Mg[Cl.sub.2], 50 mM Tris-HCl (pH 8.3), 75 mM KC1], using 10 mM dNTPs, 10 mM DTT, 50 U M-MLV reverse transcriptase, 2[micro]g random hexamer primers and 3 [micro]g total RNA. Parallel preparations without reverse transcriptase were included for the detection of DNA contamination.
Polymerase chain reaction (PCR) and analysis of PCR products
Three micrograms of cDNA were added to the respective oligonucleotide primers (1 nM each) and Taq polymerase (2.5 U/ml). The reaction volume was adjusted to 50 [micro]l using PCR buffer to reach the final concentrations of 10 mM Tris-HCl (pH 9.0), 50 mM KC1, 0.1% Triton X-100, 2.5 mM Mg[Cl.sub.2], dNTPs (dATP, dGTP, dCTP, dTTP, 200 [micro]M each), and 5% DMSO. After an initial denaturation for 69 s at 95 [degrees]C, 36 cycles of amplification (93 [degrees]C for 55 s, 61 [degrees]C for 45 s, 72 [degrees]C for 40 s) for cytokine and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT as internal standard) cDNAs were performed followed by a 100 s step for elongation at 72 [degrees]C. Amplification cycles (30 cycles) for iNOS cDNA comprised of a 60 s step at 95 [degrees]C for denaturation, a 1-min step at 72 [degrees]C for annealing and sythesis, and a final 100 s step for elongation at 72 [degrees]C. The following primers were used: HGPRT (362 bp) sense 5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3', antisense 5'-GAG GGT AGG CTG GCC TAT AGG CT-3'; interleukin (IL)-1 (400 bp) sense 5'-GCA ACT GTT CCT GAA CTC A-3', antisense 5'-CTC GGA GCC TGT AGT GCA G-3'; IL-10 (256 bp) sense 5'-TAC CTG GTA GAA GTG ATG CC-3', antisense 5'-CAT CAT GTA TGC TTC TAT GC-3'; IL-12 (312 bp) sense 5'-CGT GCT CAT GGC TGG TGC AAA G-3', antisense 5'-CTT CAT CTG CAA GTT CTT GGG C-3'; IL-18 (440 bp) sense 5'-ACT GTA CAA CCG GAG TAA TAC GG-3', antisense 5'-AGT GAA CAT TAC AGA TTT ATC CC-3'; tumour necrosis factor (TNF)-[alpha] (276 bp) sense 5'-ATG AGC ACA GAA AGC ATG ATC-3', antisense 5'-TAC AGG CTT GTC ACT CGA ATT-3'; IFN[.sub.-[gamma]] (405 bp) sense 5'-TAC TGC CAC GGC ACA GTC ATT GAA-3', antisense 5'-GCA GCG ACT CCT TTT CCG CTT CCT-3'; IFN[.sub.-[alpha]] (692 bp) sense 5'-AAC AGC CCA GAG GAC AAA CAG CAT CTT CAA G-3', antisense 5'-AAA TCA TGC ACA AAT GAC TGA TAT TTT TG-3'; iNOS (798 bp) sense 5'-CAT GGC TTG CCC CTG GAA GTT TCT CTT CAA AG-3', antisense 5'-GCA GCA TCC CCT CTG ATG GTG CCA TCG-3'. For analysis of PCR products, 8 [micro]l aliquots from each reaction were electrophoresed in a 1.5% agarose gel containing 0.2 [micro]g/ml ethidium bromide.
PCR reactions were set up as described above. Once each reverse transcription-polymerase chain reaction (RT-PCR) reaction had been terminated, the PCR products were electrophoresed (vide supra) and their relative amounts determined by densitrometric analysis using a FLA-2000G IP/Fluorescent image analyser (Fujifilm Ltd., Tokyo, Japan) according to the instructions of the manufacturer. Gels were dried, deposited on the glass side of the FLUOR STAGE in the main unit's Stage Loading Unit, and exposed to the excitation light source at 473 nm. Fluorescent light was detected using the light-receiving filter O580. The intensities of the bands were scanned (Image Reader 1.5; Fujifilm Ltd., Tokyo, Japan; Image Gauge 3.0 software). The RT-PCR levels of the transcripts were expressed as a percentage of the internal standard, HGPRT, defined as 100%.
Results and discussion
In recent studies we have reported immune modulatory effects of P. sidoides root extracts and their constituents including the release of nitric oxides (iNO) and TNF, IFN-like activities, the stimulation of IFN-[.sub.-[beta]] synthesis and the increase of natural killer cells activity (Kayser et al., 2001; Kolodziej et al., 2003). Functional assays, however, do not provide any information regarding the underlying molecular mechanisms. To gain insight into the mode of action of the demonstrated immune modulatory activities of Eps[R] 7630 at the molecular level, we embarked on a set of experiments for the expression of transcripts of iNOS and the cytokines IL-1, IL-10, IL-12, IL-18, IFN-[.sub.-[alpha]], IFN-[.sub.-[gamma]] and TNF-[.sub.-[alpha]] using RT-PCR.
Conspicuously, the Leishmania infection of RAW 264.7 cells per se induced the expression of IL-1 and TNF-[.sub.-[alpha]] transcripts, which may be seen in the light of a stimulated moderate immune response (Fig. la). Activation of parasitised cells with the stimulus IFN-[.sub.-[gamma]] + LPS induced strongly the production of iNOS, IL-1, 12, I1-18, TNF-[.sub.-[alpha]], and IFN-[.sub.-[alpha]] mRNAs, while these mRNA levels were expressed less prominently in non-infected cells (Fig. 1b).
With the gene expression profiles of untreated and IFN-[.sub.-[gamma]]+LPS treated RAW 264.7 cells in non-infected and parasitised conditions in mind, the effects of Eps[R] 7630 and some constituents, gallic acid and umckalin, on the aforementioned mRNA expressions were analysed. As shown (Fig. 1c), Eps[R] 7630 (50 [micro]g/ml) was capable of significantly enhancing the iNOS and cytokine mRNA levels in infected cells when compared with those in non-infected conditions at 4h after incubation, when stimulation is in general maximal (Radtke et al., 2004). This finding lends support to the previously demonstrated NO- and TNF-inducing potentials as well as IFN-like activities of P. sidoides extracts and their constituents at functional levels (Kayser et al., 2001; Kolodziej et al., 2003). That this stimulatory effect of Eps[R] 7630 on gene expressions and macrophage activation was conspicuously evident in just infected cells may be of special benefit, indicating that the sensitised non-specific immune system reacts more effectively when needed.
A remarkable feature of the expression profile induced by the P. sidoides extract, and also in contrast to activation by IFN-[.sub.-[gamma]] + LPS was the production of IFN-[.sub.-[gamma]] transcripts. Although it is known that macrophage functions are intimately related to the IFN system, these cells are commonly considered to produce type I IFN and to represent only targets for IFN-[.sub.-[gamma]]-induced activation (Mogensen and Virelizier, 1987). The expression of IFN-[.sub.-[gamma]] itself in cells of monocytic lineage has merely been noted under certain physiological and pathological conditions (Gessani and Belardelli, 1998). Since respiratory tract infections are frequently caused by viruses, the modulation of the IFN system endowed with potent immunomodulatory functions (Biron, 2001; Gessani and Belardelli, 1998) may significantly contribute to the beneficial effects in Eps[R] 7630 therapy. The detection of Eps[R] 7630 and gallic acid (vide infra) as IFN-[.sub.-[gamma]]-inducing extract and plant constituent, respectively, prompted a closer look to polyphenols regarding this particular inducing capability (Kolodziej and Kiderlen, 2005). This study clearly showed that the IFN-[.sub.-[gamma]] mRNA expression appears to be an uncommon feature of herbal preparations and secondary products.
[FIGURE 1 OMITTED]
Also worthy of mention is the up-regulation of IL-12 and IL-18 mRNA levels in that both cytokines are critical to host defence against a variety of (intracellular) pathogens. (Trinchieri, 1995, 2003; Sugawara, 2000).
In search of the inducing principle, the extract was suspended in MeOH to afford a MIF and a MSF, respectively. The residues of both phases were again subjected to gene expression analyses (Figs. ld and e). Conspicuously, the gene expression profiles induced by MIF and MSF paralleled those of Eps[R] 7630 (Fig. 1c) and the negative control (Fig. 1a), respectively, indicating that the underlying principle of the observed mRNA up-regulations in infected RAW 264.7 cells was apparently present principally in the former fraction. This finding does not necessarily imply that the MeOH-soluble portion is devoid of any pharmacological activities contributing to the documented beneficial effects of Eps[R] 7630. At present, both MIF and MSF are the subject of detailed compositional studies.
In the absence of comprehensive chemical information, the readily available gallic acid, though a minor constituent, and umckalin, were analysed for their gene expression inducing potentials. Of these, only gallic acid induced pronounced iNOS and cytokine mRNA levels including the notable expression of IFN-[.sub.-[gamma]] transcripts in parasitised RAW 264.7 cells, reminiscent of the expression profiles of both Eps[R] 7630 and MIF. Although this result identified gallic acid as a IFN-y gene-inducing factor, the relatively high sample concentration of gallic acid required and its low amount in the Eps[R] 7630 extract suggests the presence of a hitherto unknown co-occurring gene activating principle in this therapeutically used product. Thus, the contribution of gallic acid to the overall effect appears less important. Although umckalin, a chemical marker of P. sidoides (Latte et al., 2000), represents one of the potent coumarins exhibiting NO-releasing and IFN-like activities, this metabolite was found to represent a poor inducer of cytokine gene expressions, at least in this experiment.
In conclusion, we have demonstrated that Eps[R] 7630 induced strongly the gene expressions of iNOS and a series of cytokine mRNAs, thus providing for the first time convincing support for the established immunomodulatory actions of this herbal remedy at the molecular level. The remarkable gene expressions in just infectious conditions suggest that Eps[R] 7630 only mediates this immunomodulatory effect in the presence of an infectious agent, e.g., when needed. Clearly, further studies are needed to unravel exactly the cellular and molecular mechanisms that underlie the various pharmacological actions of Eps[R] 7630.
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W. Trun (a), A.F. Kiderlen (b), H. Kolodziej (a,*)
(a) Institut fur Pharmazie, Pharmazeutische Biologie, Freie Universitat Berlin, Konigin-Luise-Str. 2+4, D-14195 Berlin, Germany
(b) Abteilung Infektionskrankheiten, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany
Received 14 June 2005; accepted 4 July 2005
*Corresponding author. Tel.: + 30 838 53731; fax: + 30 838 53729.
E-mail address: firstname.lastname@example.org (H. Kolodziej).