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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 316-321, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0106OC

Intratracheal Macrophage-Activating Lipopeptide-2 Reduces Metastasis in the Rat Lung

Kiyoshi Shingu, Carsten Kruschinski, Anke Lührmann, Karsten Grote, Thomas Tschernig, Stephan von Hörsten and Reinhard Pabst

Departments of Functional and Applied Anatomy, and of Cardiology and Angiology, Medical School of Hannover, Hannover, Germany

Address correspondence to: Carsten Kruschinski, M.D., Functional and Applied Anatomy 4120, Medical School of Hannover, D-30625 Hannover, Germany. E-mail: Kruschinski.Carsten{at}MH-Hannover.DE


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Primary surgery of tumors bears the risk of metastasis to organs such as the lungs. In order to prevent such metastatic processes, in the present study, local intratracheal instillation of macrophage-activating lipopeptide-2 (MALP-2) as a bacterial-derived immunomodulator of cellular host defense responses was performed, and the effects on tumor cell clearance as well as tumor colonization were investigated in the lungs of Fischer 344 (F344) rats. Compared with vehicle controls, local administration of MALP-2 parallel to intravenous inoculation of MADB106 mammary adenocarcinoma tumor cells resulted in a significant reduction of lung colony numbers, whereas MALP-2 application 1 or 3 d afterwards was not effective. Quantification of leukocyte subsets in the lung tissue by immunohistochemistry revealed a significant increase of the number of monocytes in situ, as well as an increased co-localization of Natural Killer (NK) cells with tumor cells. Synthetic MALP-2 is easily available, with virtually no limitation to the amount of compound, and easily applicable by inhalation. Therefore, as local immunostimulative effects of the bacterial antigen MALP-2 have successfully been demonstrated, its use as an immunotherapeutic agent is worth further investigation.

Abbreviations: alkaline phosphatase/anti–alkaline phosphatase, APAAP • 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester, CFSE • Fischer 344 rats, F344 rats • interleukin, IL • macrophage-activating lipopeptide-2, MALP-2 • monocyte chemotactic protein, MCP • macrophage inflammatory protein, MIP • nuclear factor-{kappa}B, NF-{kappa}B • Natural Killer, NK • reverse transcriptase–polymerase chain reaction, RT-PCR • Toll-like receptor, TLR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has recently been shown that topical administration of cytokines such as interleukin (IL)-2 in lung metastasis is of clinical value due to less systemic side effects and quality-of-life-benefits for the patient. A significant increased overall survival in nearly 200 cases of pulmonary metastases in renal cell carcinoma has been reported (1). It is known that primary tumor surgery bears the risk of cancer cell dissemination due to the operative procedure at the tumor site (2). Detection of the mRNA of selected genes specific for certain tumors has successfully been performed by reverse transcriptase–polymerase chain reaction (RT-PCR) of intraoperatively-gained periphery blood samples, for instance in breast (3), prostate (4), and colorectal cancer (5). Therefore, prevention of metastasis to lungs during primary tumor surgery by a local stimulation of the immune system of the lungs might be another prophylactic approach, as the time point of metastasis can be assessed so that immunocompetent cells can be stimulated in due time.

For this purpose, we considered the topical application of the 2-kD macrophage-activating lipopeptide-2 natural stereoisomer (MALP-2) that has originally been isolated from a clone of Mycoplasma fermentans (6). It has been shown to induce chemoattractant proteins such as macrophage inflammatory protein (MIP)-1{alpha}, monocyte chemoattractant protein 1 (MCP-1), and MIP-2 and to promote leukocyte infiltration (7). A certain amount is known about the signal pathways or the cell-surface receptors for MALP-2 (8). A new class of receptors of the innate immune system, the so-called Toll-like receptors (TLRs), was recently discovered (9), and the MALP-2–mediated response depends on the activation of TLR-2 and -6, which work cooperatively in the recognition of MALP-2 (8, 10). The TLR family, whose cytoplasmic domain is homologous to that of IL-1R, has been shown to interact with an adapter molecule, MyD88, for the activation of IL-1R–associated kinase (IRAK) (11). Ultimately, nuclear factor (NF)-{kappa}B translocates from the cytoplasm to the nucleus and activates genes with NF-{kappa}B binding sites in their promotors (12, 13). Due to its immunostimulatory effects on the monocyte/macrophage system, MALP-2 seems to be a useful mediator to activate early host defense mechanisms against tumor cells. An influx of neutrophils in the bronchoalveolar lavage of rat lungs at 24 h after intratracheal MALP-2 administration, followed by macrophages on Days 2 and 3, has recently been shown (14). As a very rapid recruitment of different leukocyte subsets has been demonstrated already minutes after a challenge by metastatic cells (15), the question for the present study was whether MALP-2 would even potentiate such effects and thus contribute to a more effective tumor cell killing.

Therefore, in the present study, an established rat model of metastasis was used (15) to investigate whether intratracheal MALP-2 instillation immediately followed by intravenous tumor cell injection, mimicking intraoperative dissemination of metastatic cells, would inhibit lung metastasis. In addition, lungs were examined immunohistochemically in order to identify changes of certain leukocyte subsets in response to MALP-2 treatment. In this study, a synthetically available bacterial antigen was used as prophylactic agent, showing a significant induction of leukocyte tumor cell interactions as well as a reduction of lung surface metastases. It represents a novel approach instigating further investigations to confirm its efficacy in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, Injection of Tumor Cells, and Processing of Lungs
F344 rats were obtained from a breeding colony kept in barrier-reared conditions at the Central Animal Laboratory at the Medical School of Hannover, Germany. Rats were kept in a specific pathogen-free facility at 25°C under a 12-h light/12-h dark cycle (lights on at 7:00 A.M.), with ad libitum access to food and water. For the experiments age-matched 8-wk-old male rats were used. All research and animal care procedures were approved by the Lower Saxony district government (Hannover, Germany) and followed principles described in the European Community's Council Directive of November 24, 1986 (86/609/EEC).

Cell culture, injection of tumor cells, dissection of the animals, and immunohistochemistry were conducted as previously described (16). In brief, 1 x 106 MADB106 tumor cells (clone kindly provided by Prof. S. Ben-Eliyahu, Tel Aviv, Israel) derived from log phase of tumor growth were injected via the lateral tail vein (intravenously), and lungs were removed at different time points thereafter. The 9-10 dimethyl-1-2-benzanthracene-induced MADB106 mammary adenocarcinoma syngeneic tumor is a selected variant cell line obtained from a pulmonary metastasis produced by the intravenous injection of the MADB100 parental adenocarcinoma induced in F344 rats (17). As the tumor is originally derived from the metastatic tissue itself, the potential to seed in the lungs can be anticipated and is furthermore supported by previous experiments (18). For in situ quantification of tumor cells at 60 min after injection, cells were vital dye stained using the fluorescein derivate 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) before injection (18). For quantification of lung surface colonies at later time points (3 wk after tumor cell inoculation), en bloc dissected lungs and the heart were injected with 8 ml Bouin's solution (72% saturated picric acid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixed in the same solution until subpleural lung nodules were counted. The time-table of injection and harvesting of the lungs in the different experiments is shown in Figure 1.



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  		Figure 1. Experimental design. Harvesting of the lungs for immunohistochemistry was performed 1 h after parallel administration of MALP-2 and tumor cells (A). For the second set of experiments, the counting of subpleural lung surface colonies, MALP-2 was applied simultaneously (B), and 1 (C) or 3 d (D) after tumor cell inoculation. In these cases, the lungs were collected 21 d afterwards.

 
Intratracheal Administration of MALP-2
MALP-2 was synthesized and kept as a stock solution of 1 mg/ml in water-2-propanol (1:1) (vol/vol) at 4°C (6). The exact peptide content was determined by amino acid analysis. For in vivo use, MALP-2 was diluted with isotonic saline for injection (Fresenius, Bad Homburg, Germany). The rats were anesthetized by isoflurane and placed into a vertical position with the heads upwards. A blunt cannula was inserted into the trachea via the mouth, and 1% MALP-2 solution (250 µl/rat) or an equal volume of saline (sham control) was instilled intratracheally, followed by 1 ml air. The animals were held in this position until they recovered from the anesthesia. MALP-2 was instilled at three different time points; i.e., simultaneously with tumor inoculation and 1 or 3 d afterwards.

Immunohistochemistry of Leukocyte Subsets and CFSE-Labeled Tumor Cells in Lungs
Analysis of leukocyte subsets was performed using monoclonal antibodies (mAbs) characterizing granulocytes (mAb RP1), NK cells (mAb 3.2.3), monocytes (mAb ED1), CD4+ T-cells (mAbs R73+W3/25+), and CD8+ T-cells (mAbs R73+OX8+). The characterization of these mAbs has previously been summarized (19–21). Identification of CFSE-labeled MADB106 tumor cells was achieved using mAb against the intracellular CFSE antigen (anti-fluorescein isothiocyanate [FITC] mAb DE1, mouse, 1:100; Boehringer, Mannheim, Germany).

For immunohistochemistry, two consecutive stainings with the alkaline-phosphatase/anti–alkaline-phosphatase (APAAP) complex were performed (18). In brief, 6 µm cryostat sections were incubated with the primary anti–CFSE-mAb (DE1) for 60 min at room temperature. Sections were washed with TBS-Tween followed by incubation for 45 min with the bridging antibody (100 µl DAKO Z 0259, 1/50 in 5% rat serum in PBS-Tween, rabbit anti-mouse; DAKO, Hamburg, Germany) diluted in 5% rat serum. After another rinse, the APAAP complex (100 µl DAKO D 0651, 1/50 in TBS-Tween, mouse) was added and the sections were again incubated for 45 min followed by addition of the substrate Fast Blue (Sigma, Deisenhofen, Germany) for 25 min. The incubation with the primary mAb was performed for 60 min at room temperature, followed by an identical procedure except for Fast Red (Sigma) being the substrate. Finally, sections were counterstained with Mayer's hemalum.

All primary antibodies were obtained from the same host. Cross-reactivity was avoided because the first single staining of the tumor cells was completed, including developing of the substrate, which blocked free arms of the bridging antibody. Then, the second staining of the leukocytes followed. However, cross-reactivity could also be excluded, because no double-positive cells were observed and because the leukocytes and the tumor targets have a different morphology. In addition, isotype controls were performed, as well as control sections in which one or both primary antibodies were omitted.

Quantification of CFSE-Labeled Tumor Cells and Leukocyte Subsets as well as Number of Co-Localizations between Both Cell Types
Lungs were collected at 60 min after the intratracheal MALP-2 or sham instillation followed by the intravenous inoculation of 1 x 106 MADB106 tumor cells. CFSE-labeling of MADB106 cells allows the quantification of tumor cells in lung tissue in situ (18). In our previous studies, using a stereology-based counting technique resulted in absolute numbers of vital dye stained MADB106 cells in thick lung sections (50 µm), which significantly correlated with relative counts of cells per lung section (unpublished data). Therefore, in the present study, the assessment of DE1+ tumor cells and effector cells in lung tissue was carried out using image analysis. All CFSE-labeled MADB106 tumor cells and leukocyte subsets within a grid on the ocular lens were counted, as well as each co-localization between tumor cells and leukocytes of each subtype (Zeiss Kpl-W 12.5x; grid 0.75 x 0.75 mm, using a Zeiss Neofluar lens, x10, NA = 0.3; Zeiss, Jena, Germany). Each right upper lobe of the lungs was sectioned at six randomly chosen nonadjacent levels, on which at least 100 grid numbers were examined per lung. Co-localizations, summarized as "% conjugate formation," were defined as number of leukocytes bound to target cells divided by total number of leukocytes (22). In our experience, co-localizations are an appropriate tool to reliably quantify cells that are morphologically in close proximity in situ and most probably interacting (18). Statistically significant dynamic changes in the number of co-localizations suggest that indeed a specific immune regulatory process takes place (15).

Quantification of Lung Metastasis
Counting was conducted according to the method of Wexler (23). Subpleural lung surface colonies were identified due to a light, white appearance (Figure 2). Three weeks after inoculation, surface metastases are 1–8 mm3, mushroom-shaped, distinctly separated, and raised above the lung surface. Visible surface metastases were quantified on randomly selected lung surface areas using a gauge (1.0 cm2). Three areas per animal were examined, and subpleural lung surface colony numbers were expressed as mean/cm2.



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  		Figure 2. For immunohistochemistry, the right upper lobe (dark grey) of the rat lungs was used (A). At 3 wk, the counting of subpleural lung surface colonies was performed in a second set of experiments. At that time, surface metastases (arrow) are 1–8 mm3, mushroom-shaped, distinctly separated, and raised above the lung surface (B).

 
Quantification of TLR2 Expression Using RT-PCR
For quantification of TLR2 expression by RT-PCR, 2 x 106 rat bronchoalveolar lavage cells were isolated and stimulated with MALP (10 ng/ml). Cells were harvested at 0.5, 1, and 3 h. Total RNA from alveolar macrophages was isolated using the TriFast Reagent system (PEQLAB, Erlangen, Germany) according to the manufacturer's recommendations.

For RT-PCR, 1 µg of total RNA was reverse transcriptased (42°C for 50 min) using Superscript reverse transcriptase (Gibco/BRL, Gaithersburg, MD), oligo(dT)primers, and desoxynucleoside triphosphates. The RT product was diluted 1:5, and 5 µl were amplified using Taq DNA polymerase (1 U/reaction; Gibco/BRL). PCR was carried out for 24 cycles (GAPDH) and for 31 cycles (TLR2) in a DNA thermal cycler (Personal Cycler; Biometra, Göttingen, Germany), with initial template denaturation at 94°C for 10 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. Amplification was completed by final incubation at 72°C for 10 min. Primer pairs for GAPDH (528 bp) and TLR2 (956 bp) were purchased from MWG-BIOTECH AG (Ebersberg, Germany). The following primers were used: for GAPDH, forward primer 5'-ACCACCATGGAGAAGGCTGG-3' and reverse primer 5'-CTCAGTGTAGCCCAGGATGC; for TLR2, forward primer 5'-TGTCAGTGGCCAGAAAAGATG-3' and reverse primer 5'-GCAGAAGCGCTGGGGAATGGC-3'. PCR products were separated on 1% agarose gels containing 1x Tris-acetic acid-EDTA and ethidium bromide. Gels were analyzed by densitometry (Gel Doc 2000; Bio-Rad, Hercules, CA) using Bio-Rad Quantity One-software.

Statistical Analysis
Data obtained for the numbers of leukocytes and tumor cells or colony numbers were analyzed by one-way ANOVA, with Fisher's PLSD as the post hoc test for group differences (Statview 5.0; SAS Institute Inc., Cary, NC). Significant group effects or interactions are given in the text of the RESULTS section. Data were then further analyzed by one factor ANOVAs split by time followed by Fisher's PLSD post hoc test, if appropriate. Asterisks in the figures indicate significant post hoc effects versus controls. All data are presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Intratracheal MALP-2 Administration on Lung Metastasis in F344 Rats
Lung colonization depends on the time point of MALP-2 instillation (Figure 3). A significant effect of treatment was seen when MALP-2 was administered simultaneously with tumor cell inoculation, which resulted in a 44% reduction of lung metastasis compared with sham-injected animals (F[1.9] = 30.0; P = 0.0006; Figure 3A). In contrast, no effects on lung colonization of tumor cells were found when MALP-2 had been given 1 or 3 d after tumor cell injection (Figures 3B and 3C).



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  		Figure 3. Metastatic disease in rats topically treated with MALP-2 at different time points after tumor cell inoculation. Each symbol represents one rat. Number of subpleural metastases of the lung tissue per cm2 at 3 wk after intratracheal administration of 250 µl of 1% MALP-2 simultaneous with injection of 1 x 106 MADB106 tumor cells that had been CFSE labeled before for later immunohistochemical staining (A). Saline was used in the control animals. The horizontal line represents mean value. Administration of MALP-2 1 (B) and 3 d after tumor cell inoculation (C). N.B.: The MADB tumor model is especially known to be dependent on stress and the adrenergic hormone axis (35). The variability in the controls is most likely due to a differential endogenous stress response in these rats. However, when MALP-2 is administered, additional immunomodulatory effects might interfere with and even overrule these stress-dependent effects.

 
Immunohistologic Quantification of Different Leukocyte Subsets by Image Analysis
Quantification of tumor cells, as well as different leukocyte subsets in lungs of animals treated with MALP-2 at the time of tumor inoculation compared with sham-injected rats, is shown in Figure 4. Sixty minutes after drug administration, monocyte numbers in MALP-2–pretreated rats increased significantly in comparison with the control group (F[1.8] = 20.1; P = 0.003). However, one factor ANOVA did not show significant effects of treatment on either tumor cell numbers (Figure 4A) or numbers of granulocytes, NK cells, and T-cells (CD4+, CD8+) (Figure 4B). These results suggest that MALP-2 rapidly attracts monocytes in vivo, whereas other cell types remain unaffected by the macrophage-activating lipopeptide.



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  		Figure 4. Immunohistochemical quantification of tumor cells, effector cells, and their co-localizations. (A) The mean number of CFSE-labeled tumor cells per grid in immunohistologic APAAP staining using the mAb DE1. The specimens were gained 60 min after tumor cell/MALP-2 administration. Data represent mean value ± SEM. (B) Mean number of leukocyte subpopulations per grid in immunohistologic staining of lungs 60 min after application of MALP-2 simultaneous with tumor cell inoculation. The different leukocytes were labeled by an APAAP staining using the following antibodies: mAb RP1 (granulocytes), mAb 3.2.3 (NK cells), mAb ED1 (monocytes), mAbs R73+W3/25+ (CD4+ T-cells), mAbs R73+OX8+ (CD8+ T-cells). Data represent mean value ± SEM. (C) Conjugate formation (%) of different leukocyte subsets with tumor cells. Number of leukocytes bound to target cells related to total number of leukocytes per grid (co-localizations). Data represent mean value ± SEM. Open bars, control; solid bars, MALP-2.

 
Co-Localization of Tumor Cells with Different Leukocyte Subtypes
The microscopic findings of CFSE-labeled MADB106 tumor cells co-localized with different leukocyte subsets (granulocytes, NK cells, monocytes, CD4+ T cells, and CD8+ T cells) at 60 min after intratracheal MALP-2 or sham administration immediately followed by intravenous inoculation of 1 x 106 MADB106 tumor cells are illustrated in Figure 5 as an example for monocytes and NK cells. Visualization of CFSE labeling by DE1-staining appears as cytoplasmic and cell membrane–associated highly sensitive and specific stain with virtually no background. Under these in situ conditions, co-localization of effector with target cells is frequently observed in either MALP-2 or sham-instilled lungs, as shown in Figure 5.



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  		Figure 5. Photomicrograph of a double-immunostained and hematoxylin-counterstained cryostat section of rat lung tissue 60 min after intratracheal administration of MALP-2 simultaneous with intravenous injection of 1 x 106 vital dye CFSE labeled MADB106 tumor cells (blue). A co-localization between tumor cell and monocyte (red) is depicted (A). Co-localizing NK cells (red) are shown in B. Magnification: x400.

 
Co-localizations were quantified using the image analysis counting technique (Figure 4C). A significant increase has only been found for NK cells being in close vicinity to CFSE-labeled cells (F[1.8] = 18.5; P = 0.004).

MALP-2–Dependent Expression of TLR2 mRNA in Alveolar Macrophages Obtained from Rat Lungs
Expression of TLR2 mRNA in bronchoalveolar cells which consisted of up to 99% alveolar macrophages isolated from rat lungs was determined by RT-PCR after in vitro stimulation of the cells with 10 ng/ml MALP-2 (Figure 6). The mRNA was markedly increased 30 min after MALP-2 stimulation (2.8-fold compared with control levels), and still elevated 1 h after stimulation (1.4-fold).



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  		Figure 6. RT-PCR. Alveolar macrophages were stimulated with 10 ng/ml MALP-2 for the indicated period of time. Total RNA was isolated and TLR2 mRNA was determined by semiquantitative RT-PCR. There was a 2.8-fold induction of TLR2 mRNA 0.5 h after MALP-2 challenge.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, a protective effect of topical MALP-2 application on lung metastasis of MADB106 adenocarcinoma cells has been demonstrated. Already 60 min after MALP-2, significant changes of leukocyte subsets in the lung tissue were found with an increased number of monocytes and more frequent co-localizations of NK with tumor cells in situ. Thus, a very early stimulation of local host defense mechanisms against tumor cells by the bacterial antigen MALP-2 appears to be a possible approach in anti-tumor therapy or as an adjuvant during intraoperative dissemination. Other preclinical and clinical studies (1) have recently demonstrated promising results with the use of cytokines such as IL-2 (1, 24) or granulocyte-macrophage colony-stimulating factor (25) in lung metastases of different primaries. In these investigations, inhalative treatment has proven to be an appropriate application form, minimizing side effects while reaching higher dosages at the site of the tumor (26). Furthermore, MALP-2 is now synthetically available, with virtually no limitation to the amount of compound.

NK cells and monocytes are both known to play an important role in host defense mechanisms against tumors (15, 26, 27). As has been demonstrated recently, the number of NK cells increases in situ within minutes after tumor cell spreading to the lungs, immediately followed by monocyte recruitment after tumor cell inoculation using the MADB106 adenocarcinoma tumor model. However, when NK cells were depleted, the recruitment of monocytes was absent, and the number of tumor cells retained in the lungs dramatically increased (15). In the present study, 60 min after MALP-2 application the number of monocytes was higher than in the control group, and the number of co-localizations between NK cells and tumor cells also significantly increased. Previously, MALP-2 inducing the neutrophil-attracting CXC chemokines IL-8 and GRO-{alpha} in human monocytes, as well as the mononuclear leukocyte-attracting CC chemokines MCP-1, MIP-1{alpha} and MIP-1ß, has been demonstrated in vitro (7). Thus, MALP-2 now also seems to promote the activation of monocytes in vivo at the site of the tumor. In a preceding study, a significant recruitment of neutrophils after 24 h, and of macrophages after 2 and 3 d, had already been shown as effects of intratracheal MALP-2 application alone (14). According to the present results, this macrophage-attracting effect occurs much earlier and can promote the activation of the innate immune system against tumor cells, as shown recently (15).

The binding site for the MALP-2 stereoisomer has been shown to be TLR2 and -6 (8, 10). So far the following is known about this receptor family concerning their distribution in different tissues: TLR2 and -6 work cooperatively in the recognition of MALP-2, which is diacetylated at the N-terminus cysteine residue. Most other bacterial lipoproteins with a triacetylated terminus solely trigger immune responses via TLR2 (10). Here, we could show that TLR2 mRNA is upregulated under MALP-2 stimulation in monocytes gained from the BAL of rats. Because TLR2 is not expressed on CD56-positive NK cells (29), the increase of co-localizations between NK and tumor cells is most likely a secondary effect attributable to monocyte activation by MALP-2. NK cells are known to express several receptors for monocyte-derived cytokines (monokines), including IL-1, IL-10, IL-12, IL-15, and IL-18 (29). Consecutively, having been activated by monocytes, NK cells produce other immunoregulatory cytokines, such as interferon-{gamma}, tumor necrosis factor-ß (lymphotoxin), IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (30). This might explain why NK cells again rapidly attract monocytes in situ, which has been shown by Shingu and coworkers (7). It is known that NK cells control the development of various experimental tumors (17, 31). In addition, monocytes effectively kill tumor cells through both antibody-dependent and antibody-independent mechanisms (32, 33), a critical prerequisite for these functions being their cellular activation (27, 28). Interferon-{gamma}, for example, dramatically enhances the cytolytic potential of monocytes (34).

Therefore, the protective-like effect of MALP-2 instillation on tumor metastasis in the lungs most of all seems to be initiated by specific monocyte activation via TLR, which is then followed by a cascade of mutual activation, mainly between mononcytes and NK cells. The present data support, therefore, the idea that stimulation of local host defense mechanisms effectively contributes to the suppression of early lung metastasis. For the first time a bacterial molecule was used to support early innate immune mechanisms. These observations are clinically relevant, as metastatic colonization could be significantly reduced at the subpleural parts of the lung tissue. MALP-2 is easily available and applicable by inhalative administration, and is therefore an interesting agent to be further evaluated for clinical use.


   Acknowledgments
 
MALP-2 was kindly donated by P. F. Mühlradt and M. Morr. Its development has continuously been supported by the DFG (grant Mu 672/2). The authors thank S. Kuhlmann and S. Faßbender for excellent technical assistance, and S. Fryk for correction of the English.

Received in original form July 8, 2002

Received in final form August 27, 2002


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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