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(Journal of Leukocyte Biology. 2002;72:780-789.)
© 2002 by Society for Leukocyte Biology

Role of the autocrine chemokines MIP-1{alpha} and MIP-1ß in the metastatic behavior of murine T cell lymphoma

Patricia Menten*, Alessandra Saccani*, Chris Dillen*, Anja Wuyts*, Sofie Struyf*, Paul Proost*, Alberto Mantovani{dagger}, Ji Ming Wang{ddagger} and Jo Van Damme*

* Laboratory of Molecular Immunology, Rega Institute for Medical Research, Leuven, Belgium;
{dagger} Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; and
{ddagger} National Cancer Institute, National Institutes of Health, Frederick, Maryland

Correspondence: Dr. J. Van Damme, Laboratory of Molecular Immunology, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: jozef.vandamme{at}rega.kuleuven.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ESb-MP T-cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb-MP cells. Here, we demonstrate that ESb-MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) and MIP-1ß from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb-MP cells themselves produce significant amounts of MIP-1 (~200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb-MP cells, were intact MIP-1{alpha} and MIP-1ß. Pretreatment with LPS or addition of MIP-1 inhibited the in vitro migration of ESb-MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS-treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine-produced and tissue-derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.

Key Words: migration • metastasis • endotoxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines comprise a family of chemotactic cytokines that attract various leukocyte populations to sites of inflammation. Depending on the positioning of the cysteine residues, this family has been classified into CXC, CC, C, and CX3C chemokines [1 2 3 4 ]. The CXC chemokine subfamily can further be divided in ELR+ and ELR-CXC chemokines based on the presence or absence of the Glu-Leu-Arg (ELR) motif just in front of the first cysteine. In addition to their role in cell migration, chemokines also affect other biological processes, such as angiogenesis, hematopoiesis, atherosclerosis, and HIV-1 infection [1 , 5 , 6 ]. Furthermore, they play a role in tumor growth and metastasis [7 , 8 ].

The relationship between a malignant tumor and its host is very complex. The observation that tumors are frequently surrounded by or infiltrated with inflammatory cells suggests that the immune response often attempts to reject the tumor. However, it has become clear that in some circumstances, the host defence system may actually support rather than inhibit tumor growth and metastasis [7 8 9 ]. Initiation of metastasis occurs when tumor cells acquire increased motility and invade the surrounding normal tissue in response to several mediators. The invaded tissue and the metastasizing cells secrete different molecules, including chemokines, which stimulate motility or even directional migration of tumor cells [7 8 9 ]. Some malignant tumors may preferentially metastasize to particular organs. Although the precise mechanisms for such organ-specific metastasis are not elucidated yet, chemokines may be important in this process.

Chemokines can have tumor-promoting and tumor-inhibiting effects. On the one hand, chemokine production by tumor cells can cause infiltration of macrophages into tumors [10 11 12 ]. Macrophages are known to kill tumor cells in vitro, and in addition, they have been shown to secrete ELR-CXC chemokines, which inhibit angiogenesis and as a consequence, limit tumor growth. Conversely, some chemokines (e.g., ELR+CXC chemokines) can induce degranulation of neutrophils and thus stimulate the release of enzymes that dissolve the extracellular matrix and diminish cell-cell contacts, leading to an enhanced migration of tumor cells toward the vessel wall [13 ]. Moreover, as a result of their angiogenic potential, ELR+CXC chemokines can enhance the nutrient supply to the tumor as well [12 ]. Another tumor-promoting characteristic of some chemokines is their capacity to chemoattract tumor cells, leading to metastasis [9 ].

The ESb-MP cell line is an adherent variant of a highly malignant murine T-lymphoma [14 15 16 ]. When injected in vivo, these lymphoma cells preferentially metastasize to liver and kidney. Recently, it was shown that the CC chemokines monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES), produced by mesangial cells, potentially contribute to the invasion of the kidney by ESb-MP cells [14 ]. In this study, we further investigated the role of chemokines produced by the tumor cells themselves in the invading potential of these lymphoma cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and chemokines
The ESb-MP cell line, kindly provided by Dr. V. Schirrmacher (German Cancer Research Center, Heidelberg), is an adherent subclone of ESb, a highly malignant variant of a murine methylcholanthrene-derived T cell lymphoma [14 15 16 ]. The ESb-MP cell line was selected based on its plastic adherence property. ESb-MP cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker Europe, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS; Life Technologies, Paisley, Scotland) and 60 µM ß-mercaptoethanol. Recombinant murine (rm)RANTES was purchased from Peprotech (Rocky Hill, NJ), whereas rm-macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}) and MIP-1ß were obtained from R&D Systems (Abingdon, UK). Natural murine MCP-1/JE, KC, and MIP-2 were purified from supernatant from stimulated L929 fibroblastic cells [17 ], MO fibroblastic cells [18 ], and WEHI monocytic cells [18 ], respectively.

Production and purification of chemokines
Confluent monolayers (119 flasks of 175 cm2; Nunc, Roskilde, Denmark) of ESb-MP cells were induced for 48 h with 10 µg/ml lipopolysaccharide (LPS; from Escherichia coli; 0111:B4; Difco Laboratories, Detroit, MI) in serum-free DMEM, supplemented with 60 µM ß-mercaptoethanol. By means of adsorption to silicic acid (particle size 35–70 µm, pore size 100; anA; Matrex®, Amicon Inc., Beverly, MA), a simultaneous concentration and partial purification of the chemokines present in the conditioned medium were achieved. Therefore, ESb-MP supernatant was magnetically stirred with silicic acid (10 g/l) for 2 h at 4°C. After centrifugation to sediment the silicic acid, adsorbed chemokines were washed (4°C, 10 min) once with phosphate-buffered saline (PBS) and once with PBS containing 1 M NaCl. Proteins were eluted from the silicic acid by magnetically stirring in PBS containing 1.4 M NaCl and 50% ethylene glycol for 30 min at 4°C. The eluates were further concentrated by dialysis (3.5-kDa cut-off membranes; Spectra/Por, Spectrum Medical Industries Inc., Houston, TX) against 50 mM Tris/HCl, 50 mM NaCl, pH 7.4, supplemented with 15% polyethylene glycol 20,000 (Fluka Chemie AG, Buchs, Switzerland), before loading on a heparin-Sepharose column (1.6 cmx40 cm; 10 ml/h; CL-6B; Amersham Pharmacia Biotech, Uppsala, Sweden). After washing the column with the dialysis buffer, proteins were eluted with a linear NaCl gradient (0.05–2 M NaCl in 50 mM Tris/HCl, pH 7.4; 20 ml/h; 5-ml fractions). For cation-exchange fast protein liquid chromatography (FPLC), fractions derived from heparin-Sepharose affinity chromatography were dialyzed against 50 mM formate, 0.01% Tween 20, pH 4.0. Dialyzed fractions were injected on a Mono S cation-exchange column (50 mmx5 mm; Amersham Pharmacia Biotech) equilibrated with the same buffer. After washing the column with this equilibration buffer, proteins were eluted with a linear NaCl gradient (0–1 M NaCl in 50 mM formate, 0.01% Tween 20, pH 4.0; 1 ml/min; 1-ml fractions). Proteins present in FPLC fractions were purified to homogeneity by reversed-phase high performance liquid chromatography (RP-HPLC) on a 220 mm x 2.1 mm C8 Aquapore RP-300 column (Applied Biosystems Inc., Foster City, CA) equilibrated with 0.1% trifluoroacetic acid (TFA) in MilliQ (Millipore Corp., Milford, MA) water. Chemokines were eluted with an acetonitrile gradient (0–80% acetonitrile in 0.1% TFA; 0.4 ml/min; 0.4-ml fractions).

Identification of chemokines
Proteins obtained by HPLC were checked for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions on Tris/Tricine gels [19 ] and were visualized by silver staining. Relative molecular mass markers (Life Technologies) used were carbonic anhydrase [molecular weight (Mr) 31,000], ß-lactoglobulin (Mr 18,000), lysozyme (Mr 14,000), bovine trypsin inhibitor (Mr 6500), and the ß chain of insulin (Mr 3500). The NH2-terminal amino acid sequence of pure chemotactic proteins was identified by Edman degradation on a pulsed liquid-phase protein sequencer (477/120A and Procise 491 cLC, Applied Biosystems Inc.) with on-line detection of phenylthiohydantoin-amino acids. The molecular mass of the purified chemokines was determined on an electrospray ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany). Therefore, C8 RP-HPLC-purified proteins were diluted fivefold in acetonitrile/water (1/1) including 0.1% acetic acid and were applied to the mass spectrometer by direct infusion at a flow rate of 2 µl/min. Average relative molecular masses were calculated from the summation of 100 spectra, with an accuracy of ±1.0 Da.

Chemotaxis assay
Chemotactic activity for ESb-MP cells was determined in the 48-well Boyden microchamber assay (Neuro Probe, Cabin John, MD). The wells in the lower compartment of the chamber were filled with 27 µl test sample dilutions in RPMI 1640 (BioWhittaker Europe) supplemented with 1 mg/ml human serum albumin (HSA; Belgian Red Cross). All samples were tested in triplicate within a single microchamber. The wells in the upper compartment were filled with 50 µl ESb-MP cell suspension (in RPMI 1640+1 mg/ml HSA) at a concentration of 2 x 106 cells/ml. The two compartments were separated by a 5-µm pore-size polyvinylpyrrolidone-treated polycarbonate filter (Nuclepore, Pleasanton, CA). After incubation for 2 h at 37°C in humidified air containing 5% CO2, the filter was removed, and cells were fixed and stained using Hemacolor staining solutions (Merck, Darmstadt, Germany). The migrated cells were counted in 10 microscopic fields (magnification, 500x) per well. Chemotactic indices (CI) were calculated by dividing the number of migrated cells toward the chemokine by the number of cells migrated to the negative control (medium alone). For checkerboard analysis, used to measure chemokinesis, various concentrations of the chemokine were added to the cells at the time of transfer to the upper wells of the microchamber. In desensitization experiments, cells were preincubated with buffer or with various concentrations of chemokine for 10 min at 37°C before transfer to the upper wells.

Induction of chemokines in ESb-MP lymphoma cells
ESb-MP cells were subcultivated in 24-well dishes (Nunc) and grown to confluency. For induction experiments, one of the following inducers was added to the cells: LPS from E. coli (0111:B4; Difco Laboratories), conditioned medium from Staphylococcus aureus, S. aureus enterotoxin B (SEB; Sigma Chemical Co., St. Louis, MO), Sendai virus (2050 hemagglutination U/ml), the double-stranded RNA poly I-C (PIC; P-L Biochemicals Inc., Milwaukee, WI), the plant lectin concanavalin A (Con A; Calbiochem, San Diego, CA), the phorbol ester phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.), or natural, pure interleukin (IL)-1ß [17 ]. After 48 h of stimulation, cell supernatants were harvested and kept at -20°C until assay. Murine RANTES, MIP-1{alpha}, and MIP-1ß immunoreactivity (detection limits: ~0.2 ng/ml) were measured by specific sandwich enzyme-linked immunosorbent assays (ELISAs; no cross-reactivity with other chemokines; R&D Systems).

RNase protection assay
ESb-MP cells were treated with LPS from E. coli (0.1 or 10 µg/ml; 4 h) or were left untreated. After washing the cells with PBS, total RNA was extracted from LPS-treated and untreated ESb-MP cells using the guanidium isothiocyanate method as previously described [20 ]. Chemokine and chemokine receptor mRNAs were detected using the RiboQuant Multi-Probe RPA kit (template set mCR-5, Pharmingen, San Diego, CA) following the instructions of the supplier. In brief, riboprobes were [32P]-labeled and hybridized overnight with 15 µg RNA. The hybridized RNA was treated with RNase and purified according to the RiboQuant protocol. Protected RNAs were then resolved on a 5% denaturing polyacrylamide gel. The gel was adsorbed to filter paper, dried under vacuum, and exposed on a film (X-AR, Kodak, Rochester, NY) with intensifying screens at -70°C. Results were analyzed by densitometric analysis. For quantitation, chemokine and chemokine receptor values can be expressed as a percentage of housekeeping gene (L32) expression.

In vivo metastasis of ESb-MP lymphoma cells in DBA-2 mice
ESb-MP cells (stationary flasks of 75 cm2; Nunc) were grown to semi-confluency in DMEM and supplemented with 10% FCS and 60 µM ß-mercaptoethanol. The cells were incubated with 10 µg/ml LPS for 6 h (two flasks) at 37°C or were left untreated (control: two flasks). After incubation, the adherent cells were washed with PBS (3 ml/flask) to remove growth medium and LPS and were then incubated in PBS + 0.02% EDTA (2 ml/flask) at 37°C until the cells detached. Cell suspensions were centrifuged at 250 g (10 min, 4°C) and were subsequently washed with PBS (250 g, 10 min, 4°C). ESb-MP cells were resuspended in cold PBS, and 100 µl LPS-treated or untreated cell suspension (10x106 cells/ml) was injected intravenously (i.v.) or subcutaneously (s.c.) in DBA-2 mice (~8 weeks old). When one mouse within an experiment (i.v. or s.c.) showed clinical symptoms of advanced tumor progression, all animals (injected with LPS-treated or untreated cells) from that group (i.v. or s.c.) were killed. Liver and kidneys were dissected for analysis. The degree of specific organ metastasis was determined by two independent investigators (optical scores from - to +++++). In particular, the high number of liver metastasis was evaluated by a visual score for number, size, and distribution throughout the organ. For kidneys, the number of nodules was counted with a correction for size and was compared with an independent optical score as for the liver.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chemotactic response of the murine lymphoma cell line ESb-MP to various chemokines
It was previously shown that ESb-MP lymphoma cells migrate in vitro toward the murine CC chemokines MCP-1/JE and RANTES [14 ]. These chemokines, which are produced by mesangial cells, were thought to be at least in part responsible for the metastasis of the lymphoma cells toward the kidney. The possibility that ESb-MP cells could efficiently migrate in response to other chemokines as well was investigated using various murine chemokines, including the CC chemokines MIP-1{alpha} and MIP-1ß (Fig. 1 ) as well as the CXC chemokines KC, MIP-2, and granulocyte chemotactic protein-2 (GCP-2). From these experiments, it was concluded that in addition to MCP-1/JE and RANTES, the murine CC chemokines MIP-1{alpha} and MIP-1ß were also chemotactic for ESb-MP lymphoma cells. In contrast, all CXC chemokines tested (KC, MIP-2, and GCP-2) were inactive (data not shown). The minimal effective concentrations of MCP-1/JE, MIP-1{alpha}, MIP-1ß, and RANTES for chemotaxis of ESb-MP cells were ~0.3, 0.3, 1, and 10 ng/ml, respectively. The efficacies of MIP-1{alpha} and MIP-1ß were superior to RANTES and JE, as threefold higher CI were reached (Fig. 1) .



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  		Figure 1. Comparison of the chemotactic activities of murine chemokines on ESb-MP lymphoma cells. The chemotactic potencies of murine MCP-1/JE ({triangleup}), RANTES (•), MIP-1{alpha} ({lozenge}), and MIP-1ß ({blacksquare}) on ESb-MP lymphoma cells were compared in the Boyden microchamber chemotaxis test. The results are expressed as the mean chemotactic index ± SEM from three or more independent experiments.

 
Isolation of autocrine chemotactic factors from ESb-MP lymphoma cells
In addition to determining the chemotactic response of the lymphoma cells toward chemokines, it was also interesting to study the production of chemokines by ESb-MP lymphoma cells. In particular, the production of chemotactic factors active on the ESb-MP cells themselves could be interesting, as these chemokines might increase lymphoma cell motility (chemokinesis) or in contrast, desensitize these cells for migration to chemokines produced, for instance, in the kidney (JE and RANTES).

Stimulation with LPS, a common chemokine inducer, was required, as unstimulated cells did not produce detectable chemotactic activity for ESb-MP cells nor for neutrophils or THP-1 cells (data not shown). When subjected to heparin-Sepharose chromatography (Fig. 2A ) and subsequently to cation exchange FPLC (Fig. 2B) , the chemotactic activity for ESb-MP cells eluted at 0.8 M NaCl and at 0.7 M NaCl, respectively. Finally, chemotactic proteins present in the active FPLC fractions were further fractionated and purified by RP-HPLC (Fig. 3A ). Two separate peaks of chemotactic activity were recovered, one eluting at 33% acetonitrile (fraction N° 52–56) and another at 37% acetonitrile (fraction N° 67–70). As shown by SDS-PAGE (Fig. 3B) , both peaks corresponded to 8-kDa protein bands. By means of NH2-terminal sequence analysis and mass spectrometry, the proteins in the minor HPLC peak (fraction N° 54) were identified as intact (7827 Da), murine MIP-1ß(1–69; 96%) and truncated murine MIP-1ß(3–69; 4%), missing two NH2-terminal residues. The protein in the major HPLC peak (fraction N° 67) of ESb-MP chemotactic activity corresponded to intact (7884 Da) murine MIP-1{alpha}. Thus, ESb-MP cells are capable of producing autocrine chemotactic factors, namely MIP-1{alpha} and MIP-1ß after LPS stimulation. No chemotactic activity for ESb-MP cells, neutrophils or monocytes other than as a result of MIP-1{alpha} and MIP-1ß, was detected in the supernatant of stimulated ESb-MP cells. Nevertheless, about 50 ng/ml RANTES was produced by LPS-induced ESb-MP cells (data not shown). This RANTES immunoreactivity did not coelute with the chemotactic activity for ESb-MP cells (Fig. 2A) . Further purification of this immunoreactivity by FPLC and RP-HPLC revealed a 7-kDa protein band on SDS-PAGE. Subsequent NH2-terminal sequencing confirmed the presence of intact RANTES(1–68) as well as a substantial amount (25%) of truncated RANTES(3–68). As RANTES is a weak agonist for ESb-MP cells (Fig. 1) , and truncated RANTES(3–68) is a chemotaxis inhibitor [21 ], it is logical that its chemotactic activity was not recognized during purification (Fig. 2A) . Finally, induction of MIP-1{alpha}, MIP-1ß, and RANTES by LPS was also confirmed by RNase protection assay (Fig. 4 ). The results also show that unstimulated ESb-MP cells contain detectable but weak levels of mRNA for MIP-1{alpha} and MIP-1ß, whereas the level of RANTES mRNA was marginal.



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  		Figure 2. Purification of ESb-MP cell-derived chemokines by heparin-Sepharose chromatography (A) and cation-exchange chromatography (B). (A) Conditioned medium of endotoxin-stimulated ESb-MP cells was loaded on a heparin-Sepharose column at pH 7.4 and eluted with a linear NaCl gradient (- - - -; 5-ml fractions). The protein concentrations were measured by a Coomassie blue protein assay ({triangleup}). Chemotactic activities for ESb-MP cells were determined in the Boyden microchamber assay (•). RANTES immunoreactivity ({circ}) was determined using a specific ELISA. (B) Fractions (N° 7–9) derived from heparin-Sepharose chromatography were injected on a Mono S cation-exchange column at pH 4.0 and were eluted with a linear NaCl gradient (- - - -; 1-ml fractions). Absorbance was monitored at 280 nm (—) as a parameter for the protein content.

 


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  		Figure 3. Purification of ESb-MP cell-derived chemokines to homogeneity by RP-HPLC. (A) Fractions from cation-exchange chromatography, containing chemotactic activity for ESb-MP cells, were loaded on a C8 RP-HPLC column. Proteins were eluted with an acetonitrile gradient (- - -) and were detected by measuring UV absorption at 220 nm (—). Fractions were tested for their chemotactic activity on ESb-MP lymphoma cells (•). (B) Proteins eluted from the HPLC column were analyzed for purity by SDS-PAGE under reducing conditions and were stained with silver. Markers used are indicated in Materials and Methods.

 


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  		Figure 4. Induction of MIP-1{alpha}, MIP-1ß, and RANTES mRNA by LPS in ESb-MP lymphoma cells. Total RNA was purified from LPS-induced (10 µg/ml) or untreated ESb-MP lymphoma cells. Chemokine mRNA expression was investigated using the RNase protection assay (as indicated in Materials and Methods). Ltn/lymphotactin; IP-10, interferon-inducible protein 10; TCA/thymus-derived chemokine agonist; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

 
Chemokine production by ESb-MP cells upon stimulation with various inducers
As ESb-MP cells produced MIP-1 after treatment with endotoxin, it was further evaluated under which other inflammatory and infectious conditions these cells were capable of producing MIP-1. Therefore, confluent monolayers of ESb-MP cells were stimulated for 48 h with several inducers, including LPS from E. coli, SEB, conditioned medium from S. aureus, Sendai virus, the double-stranded RNA PIC, the plant lectin Con A, PMA, and the cytokine IL-1ß. Supernatants were assayed for murine MIP-1{alpha} and MIP-1ß immunoreactivity using specific ELISAs. From Figure 5 , it can be deduced that in addition to LPS, Sendai virus, PMA, and double-stranded RNA dose-dependently induced MIP-1{alpha} and MIP-1ß production by ESb-MP cells. At optimal concentrations, LPS, virus, and PMA induced high amounts (up to 200 ng/ml) of MIP-1{alpha} and MIP-1ß, whereas double-stranded RNA caused a weak production, and SEB and IL-1ß failed to induce any of these chemokines. It is interesting that Con A and conditioned medium from S. aureus induced MIP-1ß better than MIP-1{alpha}. It can be concluded that significant amounts of MIP-1{alpha} and MIP-1ß are released by ESb-MP cells during bacterial or viral infection.



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  		Figure 5. Induction of MIP-1{alpha} and MIP-1ß in ESb-MP lymphoma cells. ESb-MP cells were grown to confluency and incubated for 48 h with one of the following inducers: LPS from E. coli, conditioned medium of S. aureus, SEB, Con A, PMA, PIC, Sendai virus, or IL-1ß or were left untreated (control). The MIP-1{alpha} and MIP-1ß concentrations measured by ELISA represent the mean ± SEM of four or more independent induction experiments.

 
Influence of MIP-1 on the in vitro migration of ESb-MP lymphoma cells
The production of MIP-1{alpha} and MIP-1ß by LPS-stimulated ESb-MP cells could increase lymphoma cell motility (if chemokinetic for ESb-MP cells) or could desensitize chemokine receptors on ESb-MP cells for a subsequent challenge with an externally produced chemokine (e.g., RANTES by mesangial cells). Hence, autocrine chemokine production could influence the migration of the tumor cells toward chemokines produced in distinct organs.

First, the potential chemokinetic effect of pure, natural MIP-1{alpha} and MIP-1ß on ESb-MP cells, i.e., the stimulation of cell motility in the absence of a chemokine gradient, was investigated by checkerboard analysis. These experiments (Table 1 ) revealed that neither MIP-1{alpha} nor MIP-1ß exerted chemokinetic activity on ESb-MP cells, as no cell migration was observed when MIP-1{alpha} or MIP-1ß (0.3–30 ng/ml) was added to the ESb-MP cells in the upper compartment of the microchamber without chemokine in the lower compartment.


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  		Table 1. Checkerboard analysisa on ESb-MP Lymphoma Cells with Murine MIP-1{alpha} and MIP-1ß

 
Second, pure, natural MIP-1{alpha} and MIP-1ß were tested for their effect on the in vitro migration of ESb-MP cells to the murine mesangial cell supernatant, known to contain MCP-1/JE and RANTES, as well as to pure, natural MCP-1/JE. ESb-MP cells were therefore preincubated with various concentrations of MIP-1 before adding to the upper compartment of the microchamber (Tables 2 and 3 ). These experiments demonstrated that MIP-1{alpha} and MIP-1ß inhibited, in a dose-dependent manner, the migration of ESb-MP cells to mesangial cell-conditioned medium and to MCP-1/JE (Tables 2 and 3) . These findings are most likely the result of receptor desensitization and/or down-regulation processes. Indeed, the CC chemokine receptors CCR1 and CCR5, which are recognized by RANTES, also interact with MIP-1{alpha} and MIP-1{alpha}/MIP-1ß, respectively, and as a consequence can be desensitized or blocked for binding of RANTES (homologous desensitization) [22 23 24 ]. Conversely, the decreased in vitro chemotactic activity of the CCR2 agonist MCP-1/JE might be the result of heterologous desensitization (at the level of signal transduction) [25 , 26 ]. Taken together, the relative quantities and the type of chemokines produced by the lymphoma cells and the organs to which the cells metastasize, respectively, can determine the direction of migration of these tumor cells.


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  		Table 2. Migration of ESb-MP Lymphoma Cells Toward Murine Mesangial Cell Supernatant in the Presence of MIP-1{alpha} or MIP-1ß Added to the Test Cells

 

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  		Table 3. Migration of ESb-MP Lymphoma cellsa Toward Different Concentrations of MCP-1/JE in the Presence of MIP-1{alpha} or MIP-1ß Added to the ESb-MP Cells

 
Influence of LPS treatment on the in vitro migration of ESb-MP cells
As LPS treatment induces MIP-1 production by ESb-MP lymphoma cells and as addition of MIP-1 to the ESb-MP cells was shown to inhibit their in vitro migration to chemokines, it was examined whether LPS treatment of the lymphoma cells also resulted in a decreased migratory capacity. Indeed, when ESb-MP cells were pretreated with LPS (0.1 or 10 µg/ml) for 6 h to allow MIP-1 induction in vitro, their responses in the chemotactic assay (2 h) to MIP-1{alpha} and MIP-1ß as well as to MCP-1/JE and RANTES were strongly reduced (Fig. 6 ).



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  		Figure 6. Effect of LPS pretreatment on the in vitro migration of ESb-MP lymphoma cells. ESb-MP cells were treated with LPS (hatched bars: 0.1 µg/ml; solid bars: 10 µg/ml) for 6 h or were left untreated (open bars) before the cells were added to the upper wells of the microchamber that contained various chemokines (JE, MIP-1{alpha}, MIP-1ß, RANTES) in the lower wells. The results represent the mean chemotactic index ± SEM from three independent experiments. Significant statistic differences were calculated using the Mann-Whitney U test (*, P<0.05).

 
In addition to the production of MIP-1{alpha} and MIP-1ß by LPS-stimulated ESb-MP cells and the subsequent desensitization of the lymphoma cells, gram-negative bacterial infection could influence the migratory capacity of ESb-MP cells by means of other mechanisms as well. Indeed, it was recently shown that treatment of monocytes with LPS down-regulates the expression of several chemokine receptors, thereby decreasing the migration of these monocytes toward various chemokines, with or without affecting the mRNA level of the chemokine receptors [27 28 29 30 31 32 ]. Therefore, we examined whether such chemokine receptor down-regulation also occurred in LPS-treated ESb-MP cells. As shown in Figure 7A and 7B , the mRNA expression levels of the chemokine receptors CCR1, CCR2, and CCR5 in ESb-MP cells were not affected by treatment with 0.1 µg/ml LPS (4 h) and were only moderately affected after treatment with 10 µg/ml LPS (for CCR1: 35% reduction, for CCR2: 30% reduction, and for CCR5: 36% increase). Thus, down-regulation of the function of CC chemokine receptors in ESb-MP cells by LPS cannot be solely a result of a decreased receptor gene expression.



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  		Figure 7. Effect of LPS pretreatment on the chemokine receptor mRNA expression in ESb-MP lymphoma cells. Total RNA was purified from LPS-treated (A: 0.1 µg/ml and B: 10 µg/ml) or untreated ESb-MP lymphoma cells. The chemokine receptor mRNA expression was investigated using the RNase protection assay.

 
Influence of LPS treatment on the metastasis of ESb-MP cells
To investigate the effects of LPS pretreatment on the metastasis of ESb-MP lymphoma cells in vivo, DBA-2 mice were injected, s.c. or i.v., with 1 x 106 ESb-MP cells, pretreated or not, with 10 µg/ml LPS for 6 h. When one mouse out of a group showed clinical symptoms of tumor progression (~day 14 for i.v. injection and ~day 20 for s.c. inoculation), all animals of that group (untreated and LPS-treated) were killed. The liver and kidneys were dissected, and the degree of metastasis into these organs was determined (Fig. 8 ).



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  		Figure 8. Reduced metastasis of MIP-1 secreting ESb-MP lymphoma cells toward liver and kidney. DBA-2 mice (8 weeks old) were injected s.c. or i.v. with 1 x 106 untreated ESb-MP cells (green) or ESb-MP lymphoma cells treated with LPS (blue; 10 µg/ml for 6 h). When one animal within an experiment (i.v. or s.c.) showed clinical symptoms of advanced tumor progression, all mice within that experiment were killed. (A) Liver and kidneys were dissected for analysis. (B) The degree of metastasis into their liver and kidneys was determined (optical scores from 0 to 5+ for the liver and for each kidney). Significant statistic differences were calculated using the Mann-Whitney U test (*, P<0.05; **, P<0.01).

 
Animals injected with untreated ESb-MP lymphoma cells showed signs of tumor progression at an earlier timepoint compared with animals that received LPS-treated lymphoma cells (data not shown). In agreement with this observation, upon histological analysis, mice injected with untreated lymphoma cells showed a higher degree of metastasis toward liver and kidney compared with mice that received LPS-treated cells (Fig. 8) . These findings indicate that although the in vitro LPS treatment of ESb-MP cells was limited in time (6 h), it was effective at inhibiting in vitro chemotaxis as well as at reducing the metastatic potential of the lymphoma cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Normal cells are generally poor producers of inflammatory chemokines if not appropriately stimulated, e.g., by infectious agents. On the contrary, malignant cells often constitutively produce chemokines, thus chemoattracting different leukocyte populations toward the tumor tissue. Indeed, solid tumors not only consist of malignant cells but also of stroma, which includes new blood vessels and as mentioned, inflammatory leukocytes such as macrophages, lymphocytes, and neutrophils [7 , 8 ]. The observation that tumor tissues are frequently infiltrated with host leukocytes led to the hypothesis that the immune system of the body often attempts to reject the tumor. Macrophages, for instance, are able to kill tumor cells in vitro. They can also secrete proteins (e.g., ELR-CXC chemokines), which inhibit angiogenesis and as a consequence, limit tumor growth. Although some earlier reports indeed mention a correlation between infiltration of mononuclear cells in certain tumors and regression of those tumors, it is now clear that the host immune system can also have an opposite, tumor-promoting role, which is likely equally important as its tumor-inhibiting role. Upon activation, for instance by ELR+CXC chemokines, neutrophils degranulate rapidly, thereby releasing enzymes that dissolve the extracellular matrix and diminish cell-cell contacts. This facilitates the migration of the tumor cells toward the vessel and hence promotes metastasis. Another tumor-promoting characteristic of ELR+CXC chemokines is their angiogenic potential, which enhances the nutrient supply to the tumor [2 ]. As some chemokines can chemoattract tumor cells expressing the corresponding receptor(s), they can also directly induce metastasis of tumor cells [9 ].

In 1991, Benke & Schirrmacher [15 ] isolated a highly metastatic variant, ESb, from a chemically induced murine T cell lymphoma [14 , 16 ]. The subclone ESb-MP was isolated from parental ESb cells by adherence to plastic. When injected in vivo, ESb-MP cells metastasized into the liver and kidneys with high frequency [14 ]. Recently, it was shown that the CC chemokines MCP-1/JE and RANTES (probable murine homologues of human CCL2 and CCL5, respectively) probably contribute to the invasion of the kidney by ESb-MP lymphoma cells. Indeed, the murine kidney mesangial cell line MES-13 constitutively produced chemotactic activity for ESb-MP cells. Furthermore, upon preincubation of the mesangial cell supernatant with anti-MCP-1/JE monoclonal antibodies (mAb) or anti-RANTES mAb, the chemotactic activity for ESb-MP cells was partially inhibited [14 ]. Biochemical purification revealed that the major chemotactic activity contained in the mesangial cell supernatant was JE/MCP-1 [14 ].

In this study, we further investigated the role of chemokines in the invading potential of these lymphoma cells. We first determined the chemotactic responses of ESb-MP cells to various murine chemokines. It was shown that in addition to MCP-1/JE and RANTES, two other CC chemokines, namely MIP-1{alpha} and MIP-1ß (probable murine homologues of human CCL3L1 and CCL4, respectively), were also chemotactic (minimal effective concentration of 1 ng/ml) but not chemokinetic for ESb-MP lymphoma cells. As a consequence, MIP-1{alpha} and MIP-1ß could potentially contribute to the metastasis of ESb-MP lymphoma cells as well. It was also interesting to study the production of chemokines by ESb-MP cells, in particular the production of chemokines active on the lymphoma cells themselves. Indeed, these chemokines could possibly desensitize for a subsequent challenge with distantly produced chemokines and hence influence the metastatic capacity of the lymphoma cells. Whereas unstimulated ESb-MP cells did not produce detectable chemotactic activity to stimulate their migration, LPS-stimulated cells produced high amounts of MIP-1{alpha} and MIP-1ß (~1 µg/106 cells). A lower (~0.2 µg/106 cells) amount of RANTES was recovered, but this did not account for the chemotactic activity as a result of the low efficiency (minimal effective dose) and efficacy (maximal chemotactic index) of this chemokine for ESb-MP cells and as a result of proteolytic cleavage. However, it cannot be excluded that RANTES is equally potent as MIP-1 at desensitizing the chemokine receptors on ESb-MP cells. A small portion of purified MIP-1ß and RANTES was present as MIP-1ß(3–69) and RANTES(3–68), lacking the two NH2-terminal residues. This truncation might be a result of cleavage by CD26/dipeptidyl peptidase IV (DPP IV), a serine protease known to cleave off the NH2-terminal dipeptide from proteins with an alanine or proline at the penultimate position. Murine MIP-1{alpha} and MIP-1ß, with a penultimate proline, are potential substrates for CD26/DPP IV, and the NH2-terminal dipeptides were efficiently cleaved off by this protease (data not shown). These findings might explain the presence of processed MIP-1ß and RANTES in the supernatant of stimulated ESb-MP cells.

In addition to LPS, PMA, Sendai virus, and to a lesser extent, PIC and S. aureus-conditioned medium were shown to induce MIP-1{alpha} and MIP-1ß production in ESb-MP cells. To investigate whether the production of these chemokines by stimulated ESb-MP cells could influence their migratory capacity toward various chemokines distantly produced in the tissues [9 , 14 ], the in vivo situation was mimicked by in vitro chemotaxis experiments, performed in the Boyden microchamber. In these experiments, ESb-MP cells were preincubated with LPS (0.1 or 10 µg/ml, 6 h), and the effect of this treatment on the in vitro migration of ESb-MP cells was studied. After LPS treatment, the ESb-MP cells showed a significantly decreased chemotactic response toward the murine chemokines MIP-1{alpha}, MIP-1ß, MCP-1/JE, and RANTES. This is probably a consequence of the LPS-stimulated production of MIP-1{alpha}, MIP-1ß, and RANTES by ESb-MP cells and the subsequent receptor desensitization on the cells. Indeed, addition of MIP-1{alpha} or MIP-1ß to the ESb-MP cells inhibited the migration of these cells toward pure MCP-1/JE and toward mesangial cell-conditioned medium, known to contain MCP-1/JE and RANTES. Alternatively, if LPS treatment of the ESb-MP cells would result in down-regulation of the expression of CC chemokine receptors, as was shown for monocytes [27 , 28 ], this process could also be in part responsible for the decreased chemotactic activity of the LPS-treated lymphoma cells. RNase protection assays indicated that the mRNA expression levels of the three CC chemokine receptors present on ESb-MP cells (CCR1, CCR2, and CCR5) were only moderately decreased after stimulation of the cells with LPS. However, it is worth mentioning that LPS might also influence the amount of chemokine receptors on the cell surface by means of other mechanisms, e.g., alteration of protein translation, enhanced receptor internalization, or degradation [29 30 31 32 ].

To investigate the effects of LPS pretreatment on the metastasis of ESb-MP lymphoma cells in vivo, DBA-2 mice were injected s.c. or i.v. with LPS-treated or untreated ESb-MP cells. Our experiments demonstrated that animals injected with untreated ESb-MP lymphoma cells showed a higher degree of metastasis toward liver and kidney compared with mice that received LPS-treated cells. These findings indicate that although the LPS treatment was limited in time (6 h), it was effective at reducing the metastatic potential of the lymphoma cells. This could in part be a result of the chemokine production by the lymphoma cells. The observation that ESb-MP cells produce large amounts of MIP-1{alpha} and MIP-1ß after stimulation with LPS and that addition of MIP-1 to ESb-MP cells reduces their in vitro migratory capacity toward chemokines supports this hypothesis. However, with the help of neutralizing antibodies against MIP-1{alpha} and MIP-1ß, the role of MIP-1 in this reduced invasion of lymphoma cells after LPS pretreatment still needs to be verified. Another way to directly analyze the effects of MIP-1 on metastasis of lymphoma cells is by delivering MIP-1 to the tumor cells, e.g., through a parvoviral system as described for MCP-3 [33 ].

In addition to the reduced migratory capacity of lymphoma cells, other mechanisms are probably responsible for the decreased invasion observed after LPS treatment of the lymphoma cells. Indeed, LPS is a powerful activator of the immune system, and although the incubation with LPS was limited in time, LPS could still induce other processes (such as changes in the expression of cell adhesion molecules and production of tumor-inhibiting cytokines) that influence the behavior of tumor cells. In this respect, it is also necessary to stress that the LPS treatment of ESb-MP cells in vitro was meant to elucidate the mechanisms of lymphoma metastasis and is not to be considered as a direct method for clinical application.

Taken together, as leukocyte-derived tumor cells remain highly responsive to chemokines, these cytokines can affect their invasive potential. The existing literature on this phenomenon using in vitro and in vivo models is limited. Nevertheless, even nonleukocytic tumor cells were shown to express chemokine receptors, which could explain their metastasis into specific organs [9 , 34 ]. As a consequence, interference with the chemokine network can be of potential use in cancer therapy. The results represented here in a model system suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between tissue-derived chemokines and autocrine-produced or circulating agonists. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.


   ACKNOWLEDGEMENTS
 
This work was supported by the Fund for Scientific Research of Flanders (FWO-Vlaanderen), the Concerted Research Actions of the Regional Government of Flanders, the InterUniversity Attraction Pole initiative of the Belgian Federal Government (IUAP), and the Quality of Life Program of the European Community. P. M., A. W., S. S., and P. P. hold fellowships from the FWO-Vlaanderen. P. M. and A. S. have equally contributed to the study. The authors thank René Conings, Jean-Pierre Lenaerts, and Willy Put for technical assistance.

Received August 7, 2001; revised June 17, 2002; accepted July 1, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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