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[Cancer Research 62, 4034-4040, July 15, 2002]
© 2002 American Association for Cancer Research

Immunology

Lymphotoxin-ß Receptor Immune Interaction Promotes Tumor Growth by Inducing Angiogenesis1

Thomas Hehlgans, Benjamin Stoelcker, Peter Stopfer, Peter Müller, Grigore Cernaianu, Markus Guba, Markus Steinbauer, Sergei A. Nedospasov, Klaus Pfeffer and Daniela N. Männel2

Departments of Pathology/Tumor Immunology [T. H., B. S., P. S., P. M., D. N. M.] and Surgery [G. C., M. G., M. S.], University of Regensburg, D-93042 Regensburg, Germany; Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, D-81675 Munich, Germany [K. P.]; and Laboratory of Molecular Immunology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119991 Moscow, Russia [S. A. N.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth of solid fibrosarcoma tumors in mice was inhibited by the release of a solublelymphotoxin-ß receptor inhibitor (LTßR-immunoglobulin fusion protein) from the tumor cells. Tumor growth arrest in mice deficient in the ligand LT{alpha}1ß2 demonstrated the requirement for activation of the LTßR on the tumor cells by host cell-derived LT{alpha}1ß2. Activation of the LTßR resulted in enhanced release of macrophage inflammatory protein-2. Blocked angiogenesis was revealed in LTßR inhibitor-producing tumor nodules by immunohistochemistry and in vivo microscopy. The growth arrest of LTßR inhibitor-producing fibrosarcomas was overcome by forced MIP-2 expression in the tumor cells. Thus, LTßR activation on tumor cells by activated host lymphocytes can initiate a novel proangiogenic pathway leading to organized tumor tissue development.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The membrane-bound heterotrimer consisting of LT{alpha}1ß23 binds as a functional ligand specifically to the LTßR, a member of the TNF receptor family. Interestingly, LTßR is expressed on many cell types with the exception of lymphocytes, whereas expression of the ligand is apparently restricted to activated lymphocytes (1) . Currently the biological functions of this ligand-receptor system are not completely understood. It has been shown that signaling through the LTßR induced death in some human adenocarcinoma tumor lines (HT-29 and WiDr) in the presence of IFN-{gamma} (2) . Combined in vivo treatment of human adenocarcinoma cells (WiDr), which form solid tumors in immunocompromised mice, with an agonistic anti-LTßR antibody and human IFN-{gamma} resulted in tumor growth arrest. In addition, signaling through LTßR has been reported to induce NF{kappa}B activation and chemokine production in a cell type restricted manner (3 , 4) . Additional results have established a constitutive requirement for LT{alpha}1ß2 signaling in maintaining normal levels of secondary lymphoid tissue chemokine and B lymphocyte chemoattractant in lymphoid tissue (5) . Recent studies using genetically modified mice indicated that LTßR is critically involved in lymphoid organogenesis and in the generation of adaptive humoral immune responses (6, 7, 8) .

It is generally accepted today that tumor growth is angiogenesis-dependent and that every increment of tumor growth requires an increment of vascular growth. Tumors lacking angiogenesis remain dormant indefinitely and rapid logarithmic growth follows the acquisition of blood supply. The tumor angiogenic switch seems to be activated when the balance of angiogenic inhibitors to stimulators is shifted toward a proangiogenic milieu (9) . Potent inducers of angiogenesis have been identified, e.g., bFGF, VEGF, and angiopoietins (reviewed in Ref. 10 ). There is great interest in identifying and modulating antiangiogenic pathways and in antiangiogenic drug development for therapeutic purposes.

Here we provide evidence that activation of LTßR on fibrosarcoma tumor cells is necessary for angiogenesis and solid tumor growth. Prevention of LT{alpha}1ß2-LTßR signaling inhibited tumor angiogenesis and neovascularization, and resulted in tumor growth arrest. In addition, we show that LTßR activation on the tumor cells induced enhanced release of MIP-2, an angiogenic CXC chemokine (11) . Thus, our studies identify the interaction of activated LT{alpha}1ß2-carying lymphocytes with LTßR-expressing tumor cells as initiating event for a previously unknown proangiogenic pathway.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male 20–25 g C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany). Mice deficient for the LTßR (6) and LT{alpha}/LTß-deficient mice4 had been backcrossed more than six times to the C57BL/6 background. Experiments were performed with age- and sex-matched mice.

Tumor Cells.
The methylcholanthrene-induced fibrosarcoma (BFS-1) was generated in a female C57BL/6 mice as described (12) . The tumor cells were maintained in vitro in DMEM high glucose medium (Life Technologies, Inc., Karlsruhe, Germany) supplemented with 5% heat inactivated FCS (Life Technologies, Inc.) and 0.05 ng/ml gentamicin (PAA Laboratories, Linz, Austria). Tumor cells (1.5 x 106 in 50 µl) were inoculated i.d. on the back of mice, and tumor growth was measured as described recently (13) .

DNA Constructs and Transfectants.
The mp55TNFR-Fc and mLTßR-Fc expression constructs were generated by insertion of the extracellular domains of the mp55TNFR or the mLTßR into the Signal pIG plus vector (R&D Systems, Wiesbaden, Germany). Stable transfectants expressing the p55TNFR-Fc or LTßR-Fc fusion protein or MIP-2 were prepared by transfection of BFS-1 cells with the corresponding expression construct using N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (Roche Diagnostics, Mannheim, Germany), following the manufacturer’s instructions. Transfected cells were selected and maintained in G418 (0.8 mg/ml) or hygromycin (0.02 mg/ml; PAA Laboratories).

Flow Cytometry.
Expression of mLTßR on BFS-1 cells was detected by flow cytometry on a FACStar Plus (Becton Dickinson, San Jose, CA) using a specific rat anti-mLTßR monoclonal antibody (mLTR1C5, IgG2a) followed by a FITC-conjugated mouse antirat IgG at concentrations of 10 µg/ml or an irrelevant isotype-matched rat IgG as control. The mLTR1C5 had been generated by immunizing a rat with mLTßR-Fc fusion protein, fusing the spleen cells with SP2 myeloma cells (14) , and screening the resulting hybridoma supernatants for positive staining of LTßR-transfected Chinese hamster ovary cells.

Western Blot Analysis.
Supernatants of BFS-1 cells stably transfected with the p55TNFR-Fc or the mLTßR-Fc expression construct were incubated with 10 mg protein G-Sepharose and incubated at 4°C for 1 h. Precipitated proteins were resolved on 12% SDS-PAGE and blotted on polyvinylidene difluoride membrane (Immobilon-P, 0.45 µm; Millipore, Eschborn, Germany). The membrane was blocked with 5% dry milk in Tris-buffered saline for 1 h at ambient temperature followed by incubation with antihuman IgG1 (1 mg/ml) in incubation buffer (1% dry milk in Tris-buffered saline) for 1 h. After washing, bound antihuman IgG1 was reacted with goat antimouse-conjugated horseradish peroxidase (Sigma Biochemicals, Deisenhofen, Germany) in incubation buffer for 1 h at ambient temperature. The membrane was washed again and developed with the enhanced chemiluminescence kit (Energene, Regensburg, Germany) following the manufacturer’s instructions.

Determination of MIP-2 and VEGF.
MIP-2 and VEGF production was determined using the corresponding ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Immunohistochemical Staining.
Polyclonal antibodies to collagen type IV (Novotec, Lyon, France) were used for immunohistochemical staining of blood vessels as described recently (15) .

Determination of Cell Viability.
Cell viability of wild-type or LTßR-Fc expressing BFS-1 cells was tested by seeding the cells in 96-well plates at the indicated numbers overnight and subsequent culture in medium either with or without the agonistic anti-mLTßR monoclonal antibody (10 µg/ml) for 24 h. Cell viability was determined by adding 10 µl 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (5 mg/ml; Sigma) at 37°C for 4 h and lysing the cells by adding 70 µl of 10% SDS per well. The plates were kept at 37°C overnight, and absorbance was measured at 540 nm. Each experiment was repeated at least three times.

Intravital Microscopy.
Tumor angiogenesis was quantified in the transparent dorsal skin fold as described in detail (16 , 17) . Mice were shaved on the back, anesthetized with ketamine hydrochloride (7.5 mg/100 mg body weight; Parke, Davis & Company, München, Germany) and xylazine (2.5 mg/100 mg body weight; Bayer AG, Leverkusen, Germany), and placed on a heating pad. One pair of titanium frames was implanted in a dorsal skin fold parallel to the dorsum so as to sandwich the stretched double layer of skin. One layer of the dorsal skin was removed in a circular area of 10-mm diameter using an operation microscope to facilitate implantation. The underlying thin layer of striated skin muscle, s.c. tissue, and epidermis was sealed with a coverslip enclosed in one of the frames. Two days later the coverslip was removed and BFS-1 cells transfected with either the LTßR-Fc or the p55TNFR-Fc construct (3 x 105 cells/animal; n = 6 animals/group) were carefully placed on the upper tissue layer as a pellet and the chamber closed again. Tumor-bearing animals were monitored in regular time intervals for the development of tumor vessels (MVD = total vascular length/observation area). For intravital microscopy the mice were placed in a Plexiglas tube with the chamber extending from a longitudinal slit and immobilized on a platform. At days 2 and 6 after tumor implantation, 10 ROIs were defined and examined by intravital microscopy (8 ROIs distributed clockwise at the tumor margin and 2 ROIs in the tumor center) after relocation by a computer-controlled stepping motor system. In vivo microscopy was performed using a modified Axiotech Vario microscope (Zeiss, Oberkochen, Germany) equipped with a filter set 09 (BP 450–490, FT 510, and LP 520; Zeiss). Observations were made using x2.5 long distance, and x10 and x20 water immersion working objectives (Zeiss) resulting in a magnification of x53, x213, and x425, respectively. Observations of the window chambers were carried out in white light transillumination technique and in epi-illumination technique after injection of 0.05 ml 5% FITC dextran 500 (mW = 500,000) for contrast enhancement. Images were recorded through a CCD video camera (PCO, Kehlheim, Germany) on S-VHS tapes for later offline analysis. MVD was determined as described (18) using a modified custom-made image software (IDL).

Statistical Analysis.
Data represent one of at least three independent experiments and are shown as the mean + SD. Statistical significance of data were determined using Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To dissect the biological consequences of LTßR signaling in tumor development the growth of syngeneic LTßR-positive fibrosarcoma cells expressing a LTßR-Fc fusion protein acting as an inhibitor of LTßR activation was compared with growth of tumor cells expressing a p55TNFR-Fc fusion protein as control. Fibrosarcoma cells (BFS-1) syngeneic to C57BL/6 mice expressed LTßR on the cell surface as shown by flow cytometric analysis using a monoclonal anti-mLTßR antibody (mLTR1C5; Fig. 1aCitation ). The expression of LTßR was also confirmed at mRNA level by reverse transcription-PCR using specific primers for mLTßR (data not shown).



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  		Fig. 1. Expression of mLTßR and fusion proteins by BFS-1 tumor cells. a, flow cytometric analysis of fibrosarcoma cells (BFS-1) using a monoclonal anti-mLTßR antibody (white area) or an isotype-matched control antibody (shaded area). b, immunoprecipitation of LTßR-Fc fusion proteins from supernatants of untransfected BFS-1 cells (Lane 1), transfected with LTßR-Fc (Lane 2), or p55TNFR-Fc (Lane 3) expression constructs and subsequent detection using antihuman IgG1 antibody. c, cell viability of BFS-1 wild-type (circles) and LTßR inhibitor-expressing cells (squares) in the presence (closed symbols) or absence (open symbols) of an agonistic monoclonal anti-LTßR antibody.

 
BFS-1 cells stably transfected with cDNA of LTßR-Fc or p55TNFR-Fc expressed comparable amounts of the corresponding fusion proteins, whereas in the supernatants of nontransfected tumor cells no fusion protein was detectable by Western blot analysis (Fig. 1b)Citation . Binding of both secreted Fc fusion proteins to their corresponding ligands was verified by flow cytometric analysis in staining activated PMN1 cells, which express LT{alpha}1ß2 on their surface (1) with supernatants of BFS-1 cells secreting the LTßR inhibitor and by immunoprecipitation of biotinylated rmTNF with supernatants of BFS-1 cells secreting the p55TNFR-Fc fusion protein, respectively (data not shown). The in vitro growth characteristics of nontransfected BFS-1 cells and LTßR inhibitor-expressing BFS-1 cells were not different even in the presence of an agonistic anti-LTßR monoclonal antibody indicating that neither overexpression of LTßR inhibitor nor stimulation of the LTßR interfered with BFS-1 cell proliferation and survival in vitro (Fig. 1c)Citation .

Solid tumor growth in vivo of BFS-1 cells expressing LTßR inhibitor was inhibited compared with growth of wild-type or control p55TNFR-Fc fusion protein-expressing tumor cells (Fig. 2a)Citation . Comparable results were obtained from independent transfection experiments and also by using pool transfectants or four individual stably transfected BFS-1 clones (data not shown). To investigate the possibil ity of whether rejection of the LTßR-Fc transfectants may account for this inhibition of tumor growth the growth pattern of wild-type and transfected tumor cells in allogeneic mice was compared. Wild-type tumors, LTßR inhibitor-, and p55TNFR-Fc-expressing tumors were completely eradicated by days 9–12 after inoculation (data not shown). In contrast, LTßR inhibitor-expressing tumor cells on syngeneic mice grew to a palpable size of ~2 mm in diameter and later on showed a highly suppressed growth characteristic for >40 days (Fig. 2aCitation ; data not shown). To test whether the amount of secreted LTßR inhibitor influences the growth of BFS-1 tumor cells, mixtures of tumor cells containing increasing percentages of LTßR inhibitor-expressing BFS-1 were inoculated. Tumor growth was inhibited when 50% or more of the tumor inoculate consisted of LTßR inhibitor-expressing BFS-1 cells (Fig. 2b)Citation . The inhibitor produced from 25% of the inoculated tumor cells was only sufficient to delay tumor growth indicating that the growth inhibitory effect of LTßR inhibitor was dose dependent.



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  		Fig. 2. Tumor growth of BFS-1 transfectants. a, solid tumor growth of syngeneic fibrosarcoma cells BFS-1 ({circ}), BFS-1 cells expressing the LTßR inhibitor ({bullet}), or the p55TNFR-Fc fusion protein ({blacksquare}) in C57BL/6 mice (n = 5). b, solid tumor growth of BFS-1 cells consisting of 100% ({bullet}), 75% ({diamondsuit}), 50% ({blacktriangleup}), 25% ({blacksquare}), or 0% ({circ}) of LTßR-Fc expressing cells in C57BL/6 mice (n = 5).

 
To: (a) support the observation that activation of the LTßR is necessary for BFS-1 tumor growth; (b) clearly identify ligand specificity; and (c) additionally determine the source of LT{alpha}1ß2, wild-type BFS-1 tumor cells were inoculated into normal syngeneic C57BL/6 and into LT{alpha}/LTß-deficient mice. Comparable retardation of tumor growth was observed in LT{alpha}/LTß-deficient mice as with LTßR inhibitor-expressing BFS-1 in normal mice (Fig. 3a)Citation . This result clearly identified LT{alpha}1ß2 as a functional ligand for the LTßR activation and also determined the host as the source for the ligand. Additionally, by using reverse transcription-PCR we were able to detect mRNA for both ligand molecules LT{alpha} and LTß in explanted fibrosarcoma tumor tissue (data not shown).



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  		Fig. 3. Tumor growth in wild-type, LT{alpha}/ß-deficient, or LTßR-deficient mice. a, solid tumor growth of BFS-1 cells ({circ}), BFS-1 cells expressing the LTßR inhibitor ({bullet}) in C57BL/6 mice, and BFS-1 cells ({square}) in LT{alpha}/ß-deficient mice (n = 4). b, solid tumor growth of BFS-1 cells in C57Bl/6 mice ({circ}) and in LTßR-deficient mice ({bullet}; n = 5).

 
To identify the responsible receptor-carrying cells, LTßR-deficient mice were inoculated with wild-type BFS-1 cells. Tumor growth in such LTßR-deficient mice was indistinguishable from tumor growth in normal wild-type mice (Fig. 3b)Citation demonstrating that LTßR on the tumor cells themselves is required for unimpaired growth.

Histological examination of tissue sections from tumors on day 9 revealed no obvious difference between wild-type and LTßR inhibitor-expressing BFS-1 tumors. No significant difference was observed in numbers of infiltrating lymphocytes, cells in the mitotic phase, Tec-3 positive cells, and cells with pyknotic nuclei as a sign for dying cells (Fig. 4aCitation ; data not shown). However, staining for collagen type IV as a marker for vessel formation (15) indicated reduced vascularization in those tumors consisting of LTßR inhibitor-expressing BFS-1 cells (Fig. 4b)Citation . Numbers as well as size of newly formed vessels were reduced in LTßR inhibitor-producing BFS-1 tumor nodules.



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  		Fig. 4. Histology of BFS-1 tumors and LTßR inhibitor-transfected BFS-1 tumors. a, H&E staining of tumor tissue derived from BFS-1 cells (A) and from BFS-1 cells expressing the LTßR inhibitor (B). b, immunohistochemical staining of blood vessels in tumor tissue derived from BFS-1 cells (A) and from BFS-1 cells expressing the LTßR inhibitor (B) using collagen type IV polyclonal antibodies.

 
To follow angiogenesis in growing tumors, intravital microscopy was performed with the two tumor cell transfectants. Hereby it became clear that implantation of LTßR inhibitor-expressing BFS-1 cells led to inhibited sprouting of new vessels from the surrounding skin area into the direction of the growing tumors from days 3 to 4 on (Fig. 5a)Citation . This observation was substantiated by quantitative evaluation of the angiogenesis on day 6 (Fig. 5b)Citation .



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  		Fig. 5. Blocked angiogenesis in tumors of LTßR inhibitor-transfected BFS-1 cells. a, intravital microscopy of tumor angiogenesis on day 9 after implantation of BFS-1 cells expressing the LTßR inhibitor (A) or the p55TNFR-Fc fusion protein (B). b, quantitative evaluation of angiogenesis in tumors on day 6 after implantation of BFS-1 cells expressing the LTßR inhibitor or the p55TNFR-Fc fusion protein (* P < 0.013); bars, ±SD.

 
The supernatant of BFS-1 cells stimulated with the agonistic monoclonal anti-LTßR antibody mLTR1C5 was tested for the presence of the angiogenic mediators VEGF and MIP-2. Whereas the amount of VEGF generated into the supernatant by the tumor cells was not affected by the addition of the antibody, increased amounts of MIP-2 were released from BFS-1 cells after LTßR stimulation (Fig. 6a)Citation . Comparison of MIP-2 production ex vivo from explanted wild-type and LTßR inhibitor-expressing BFS-1 tumor tissue, respectively, on day 8 revealed a significantly higher MIP-2 expression in wild-type tumors (Fig. 6b)Citation . This finding correlated with the impaired MIP-2 production in vitro and the reduced tumor growth in vivo.



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  		Fig. 6. MIP-2 production correlates with tumor growth. a, enhanced MIP-2 release after LTßR activation. VEGF and MIP-2 levels in supernatants of BFS-1 cells 24 h after addition of a monoclonal anti-mLTßR antibody. b, MIP-2 release of explanted BFS-1 wild-type ({bullet}) or LTßR-expressing ({blacktriangleup}) tumor tissue at day 8 (P < 0.038). c, solid tumor growth of syngeneic fibrosarcoma cells expressing the LTßR inhibitor ({circ}) or cotransfected with a MIP-2 expression construct ({bullet}) in C57BL/6 mice (n = 5).

 
To additionally investigate whether MIP-2 production as a result of LTßR stimulation is important for growth of BFS-1 tumors and because preliminary experiments with systemic neutralization of MIP-2 failed, LTßR inhibitor-expressing BFS-1 cells were cotransfected with a MIP-2 expression construct. Whereas, again, growth of the LTßR inhibitor-expressing tumors was impaired, MIP-2 cotransfected tumors grew much better (Fig. 6c)Citation . Regaining of growth by MIP-2 expression clearly indicated that activation of the LTßR and ensuing up-regulation of MIP-2 expression is important for growth of BFS-1 fibrosarcoma tumors.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor cell cytotoxicity induced by LTßR activation has been described for a very limited range of tumor cells (2) . However, activation of the LTßR on BFS-1 tumor cells by an agonistic monoclonal antibody mLTR1C5 did not affect tumor cell proliferation in our experiments. The tumor cells did not show any signs of cell death when stimulated in vitro with the antibody neither in the presence nor in the absence of IFN-{gamma} (data not shown). Apart from the direct antitumoral effect other biological functions of LTßR activation have been reported as well. NF{kappa}B activation and the release of IL-8 and RANTES from A375 human melanoma cells have been described as results of LTßR stimulation (3 , 4) . Similar functional activity was ascertained in the mouse system in this report by documenting MIP-2 production from BFS-1 cells after stimulation with the agonistic antibody mLTR1C5. Also, NF{kappa}B activation was induced by mLTR1C5 in LTßR-positive BFS-1 and L929 cells (data not shown).

Our experiments with LT{alpha}/b gene-deficient mice clearly demonstrate that host cells are the source for the stimulating ligand. Because LTß, the membrane anchoring part of the heterotrimeric ligand LT{alpha}1ß2, is exclusively produced by activated lymphocytes and natural killer cells, the cellular source of LT{alpha}1ß2 needs to be determined in future experiments with tissue-restricted LT{alpha}/b gene-deficient mice. Obviously, LTßR activation is not only required for lymphoid organogenesis but also for the development of solid tumor tissue. This implies new significance for the function of this interesting member of the TNF receptor family, which stands at the interface of the immune system interacting with cellular systems indispensable for organogenesis.

The fact that activation of the LTßR can initiate cytotoxic as well as cell protective signaling pathways, depending on the cell type or the experimental conditions, is reminiscent of the signaling pathways following activation of the TNFR type 2. These two receptors are more closely related than the two TNFR with each other (19) . In addition, both receptors lack classical death domains in their intracellular domains and share a TRAF-2 binding domain, which has been found to be responsible for NF{kappa}B activation (20) . The increase of MIP-2 production after stimulation of the LTßR might be explained by the fact that the promoters of both IL-8 and MIP-2, which is the murine functional homologue of IL-8 (21) , contain binding sites for regulatory elements, e.g., NF{kappa}B, which are activated after LTßR stimulation (22 , 23) .

The observation that angiogenesis correlates with tumor malignancy is well accepted (10) . Our finding of a dose-dependent inhibitory effect of the LTßR inhibitor produced by the tumor cells fits the idea of angioneogenesis as a limiting factor for solid tumor growth. Implantation of LTßR inhibitor-expressing tumor cells on one side of the back of the mouse failed to affect wild-type tumor growth on the other side of the back demonstrating the local restriction of the action (data not shown). Enhanced MIP-2 production from the explanted growing tumor compared with LTßR inhibitor-producing tumors was demonstrated. Therefore, in our model the CXC chemokine MIP-2 could potentially attract and activate neutrophils and lymphocytes, which then might release angiogenic factors like VEGF and bFGF to induce vessel growth in the tumor microenvironment. Such support for tumor angiogenesis can become critical when tumor cells only insufficiently prepare their microenvironment directly.

Alternatively, a direct local effect of MIP-2 on endothelial cells might be envisaged as supported by data obtained in the human system. The human IL-8 is known as a multifunctional cytokine that has both angiogenic effects and potent leukocyte chemotactic and activation properties (24, 25, 26, 27) . IL-8 has been shown to stimulate chemotaxis and proliferation of human umbilical vein endothelial cells similar to bFGF, and was found to be proangiogenic in vivo (28) . In addition, IL-8 has been found in tissues of many neoplastic diseases (29, 30, 31) , and to be functional for neovascularization, both IL-8 and IL-8 receptors must be present within the tumor microenvironment (32) . The presence of IL-8 receptors on endothelial cells lining the vessels in human tumor tissue, which is critical for the recognition of IL-8 as an angiogenic factor, has been described recently (32) . Presence of IL-8 receptors on the tumor cells themselves could result in enhanced tumor cell proliferation. MIP-2, which is structurally homologous to the human CXC chemokines GRO-ß/{gamma}, represents the murine homologue of IL-8 in terms of function (21 , 33) and binds to the mouse homologue of the human IL-8 receptor (34 , 35) . It has been reported recently that like IL-8 MIP-2 is also chemotactic for endothelial cells and induces neovascularization (11) . Whether the mouse IL-8 receptor homologue is expressed on endothelial cells or on BFS-1 cells is presently not known.

The reconstitution of tumor growth in the presence of the LTßR inhibitor by MIP-2 supplementation firmly supports the idea that activation of the LTßR on the BFS-1 tumor cells by interaction with activated host lymphocytes is a prerequisite for enhanced MIP-2 production. This angiogenic chemokine might, in a paracrine manner, diffuse to the nearest blood vessel signaling, either directly or indirectly, the endothelial cells to start the angiogenic process. Very clearly, different tumor types have developed different mechanisms to induce angiogenesis. In regard to potential therapeutic intervention strategies the finding that in some, tumors angiogenesis is initiated by activated host lymphocytes activating the LTßR on tumor cells for subsequent MIP-2 production certainly deserves additional investigations.


   ACKNOWLEDGMENTS
 
We thank M. Alimzhanov for outstanding contribution to the development of LT{alpha}/LTß double-deficient mice, B. Ruhland for excellent technical assistance, Drs. R. C. Krieg and O. Gleich for photographic documentation, and Dr. D. Breitkreutz for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 S. A. N. is International Research Scholar of Howard Hughes Medical Institute. Back

2 To whom requests for reprints should be addressed, at Department of Pathology/Tumor Immunology, University of Regensburg, F.-J.-Strauss-Allee 11, D-93042 Regensburg, Germany. Phone: 49-941-944-6626; Fax: 49-941-944-6602; E-mail: daniela.maennel{at}klinik.uni-regensburg.de Back

3 The abbreviations used are: LT{alpha}1ß2, one molecule lymphotoxin-{alpha} and two molecules lymphotoxin-ß; LTßR, lymphotoxin-ß receptor; TNF, tumor necrosis factor; NF{kappa}B, nuclear factor {kappa}B; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; MIP, macrophage inflammatory protein; MVD, microvascular density; ROI, region of interest; IL, interleukin; TNFR, tumor necrosis factor receptor. Back

4 T. Plötzel, M. B. Alimzhanov, D. Kuprash, S. A. Nedospasov, and K. Pfeffer. Generation and characterization of mice with simultaneous inactivation of LT{alpha} and LTß genes, manuscript in preparation. Back

Received 12/17/01. Accepted 5/ 7/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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