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The Journal of Immunology, 1999, 163: 2809-2815.
Copyright © 1999 by The American Association of Immunologists

Mice with a Targeted Mutation in Lymphotoxin-{alpha} Exhibit Enhanced Tumor Growth and Metastasis: Impaired NK Cell Development and Recruitment1

Daisuke Ito2,*, Timothy C. Back{ddagger}, Alexander N. Shakhov*,{ddagger}, Robert H. Wiltrout{dagger} and Sergei A. Nedospasov3,*,{ddagger},§

* Laboratory of Molecular Immunoregulation and {dagger} Laboratory of Experimental Immunology, Division of Basic Sciences, and {ddagger} Intramural Research Support Program, Science Applications International Corp.-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, and § Laboratory of Molecular Immunology, Engelhardt Institute of Molecular Biology and Belozersky Institute of Physico-Chemical Biology, Moscow, Russia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice deficient in lymphotoxin (LT)-{alpha} lack peripheral lymph nodes and Peyer’s patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LT{alpha} is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LT{alpha} for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LT{alpha}-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LT{alpha}-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LT{alpha}-/- mice. Although LT{alpha}-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LT{alpha}-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LT{alpha} signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphotoxin (LT)4-{alpha} is a multifunctional cytokine structurally homologous to TNF (1, 2), which is expressed by activated lymphocytes (3) as a soluble homotrimer (LT{alpha}3) or membrane-associated heterotrimer with LTß (LT{alpha}1ß2 and LT{alpha}2ß1) (4). LT{alpha}3 shares TNF receptors p55 and p75 with TNF, whereas LT{alpha}1ß2 heterotrimers signal through LTßR, a distinct receptor of the same family (5). Previous studies revealed that LT/LTßR signaling plays an essential role in secondary lymphoid organ development and function. Specifically, LT{alpha}- and LTßR-deficient mice lack Peyer’s patches and all peripheral lymph nodes and display disrupted splenic T/B compartmentalization and germinal center formation after immunization with T cell-dependent Ags and defective Ig class switching (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Similar phenotypic alterations are observed in LTß-deficient mice, although most of these knockout mice have cervical, sacral, and mesenteric lymph nodes, and T/B areas segregate more clearly (9, 10, 16). Recent reports showed that LT/LTßR-mediated signals may be involved in the fetal/neonatal recruitment of lymphoid precursor cells to lymph nodes (17, 18) and the maintenance of follicular dendritic cell functions (19, 20). NK cells constitute an important population of lymphocytes involved in nonspecific host defense mechanisms. Although LT{alpha} is expressed by NK cells at significant levels, the contribution of LT{alpha} to NK function has not been reported. In this study, the LT{alpha}-/- mice (6) were examined for in vivo and in vitro NK function. LT{alpha}-/- mice challenged with NK cell-susceptible experimental tumors exhibited an enhanced tumor growth and formation of pulmonary metastases. NK cells from the knockout mice were defective in migratory potential to the organs and development from in vitro bone marrow (BM) culture in the presence of cytokines. Our studies revealed a significant role for LT signaling in the regulation and development of NK activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

LT{alpha}-/- mice (6) were purchased from The Jackson Laboratory (Bar Harbor, ME), additionally backcrossed to the C57BL/6 background, and bred as heterozygotes in specific pathogen-free conditions. The knockout mice and their wild-type littermate controls were genotyped by PCR and used for experiments between 6 and 12 wk of age. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).

Cell lines

The B16F10 melanoma and 3LL Lewis lung carcinoma cell lines syngeneic to C57BL/6N mice were maintained at 37°C in a 5% CO2 incubator and grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin/streptomycin, 4 mM L-glutamine, and 2 mM sodium pyruvate. The YAC-1 lymphoma cell line of A/Sn origin was maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS, antibiotics, L-glutamine, and sodium pyruvate.

Reagents and mAbs

Polyinosinic-polycytidylic acid (polyIC) and polyL-lysine stabilized in carboxymethyl cellulose (polyICLC) (21) was provided by Dr. Hilton Levy (National Cancer Institute, Bethesda, MD). Monoclonal Abs specific for mouse CD3e and NK1.1 cell surface Ags were purchased from PharMingen (San Diego, CA). Recombinant murine IL-2 and recombinant human IL-15 were purchased from PeproTech (Rocky Hill, NJ) under contract by the Biological Resources Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center.

Primary tumor growth

B16F10 cells (2 x 105) were injected s.c. into the shaved lateral flank of mice. The size of primary tumors was determined on days 14, 17, and 21 using a caliper. Tumor volume was calculated with the formula, V = (A x B2)/2, where V = volume (mm3), A = long diameter (mm), and B = short diameter (mm) (22).

Experimental metastasis

B16F10 cells (2 x 105) or 3LL cells (3 x 105) were injected i.v. into mice through the lateral tail vein. Twenty-one days later, the mice were euthanized by cervical dislocation, and lungs were harvested for metastatic colony count under a dissecting microscope.

In vitro BM culture

BM cells were obtained from the tibias and femurs of unstimulated LT{alpha}+/+ and LT{alpha}-/- mice and cultured in RPMI 1640 supplemented with 10% FBS, L-glutamine, and sodium pyruvate at 5 x 106 cells per well in 24-well plates. The cells were either unstimulated or stimulated with recombinant murine IL-2 (10 ng/ml), recombinant human IL-15 (10 ng/ml), or both. Cytotoxicity assay against YAC-1 targets was performed after 4 days in culture.

Cytotoxicity assay

Cytotoxic activity of cultured splenocytes and BM cells was assessed by standard 4-h 51Cr release assay as described previously (23). Briefly, effector cells were seeded at various concentrations in quadruplicate in 96-well round-bottom microtiter plates. YAC-1 target cells were labeled for 2 h at 37°C with 100 µCi of Na51CrO4 (Amersham Life Science, Arlington Heights, IL), and 1 x 104 labeled cells were added to the effector cells. Plates were incubated at 37°C for 4 h, and the supernatant was harvested and evaluated for levels of 51Cr with a gamma scintillation counter. The percentage of specific cytotoxicity was calculated with the formula, % specific lysis = (a – b)/(c – b) x 100, where a = experimental release (cpm), b = spontaneous release (cpm), and c = maximum release (cpm).

Isolation of lung and liver nonparenchymal cells

Nonparenchymal cells of lungs and livers were isolated as described previously (24). Briefly, lungs and livers were perfused with prewarmed HBSS. Cell suspensions from excised organs were generated with a stomacher, and cell debris was removed by passing through nylon mesh. Cells were washed and resuspended in HBSS. Nonparenchymal cells were isolated by density-gradient centrifugation with Lympholyte-M (Cedarlane Laboratories, Ontario, Canada).

Statistical analyses

The statistical significance of all assays was assessed by using the two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary tumor growth and experimental lung metastases are significantly enhanced in LT{alpha}-/- mice

In the first set of experiments, we compared primary tumor growth in LT{alpha}-/- mice and wild-type littermate controls. B16F10 melanoma cells injected s.c. into the lateral flank formed solid tumors with relatively well-defined margins in either type of mice. The tumor size was monitored on days 14, 17, and 21, and enhanced tumor growth in the LT{alpha}-/- mice was noted (p < 0.002 on days 17 and 21) (Fig. 1Go). Tumor invasion into the abdominal cavity and considerable peritoneal dissemination were observed in three of seven LT{alpha}-/- mice examined (not shown). No visible metastatic colonies were found in any organs from either knockout or control mice by day 21. These observations suggested that the local antitumor response is impaired in LT{alpha}-/- mice.



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  		FIGURE 1. Intradermal growth of B16F10 melanoma is enhanced in LT{alpha}-deficient mice. B16F10 melanoma cells (2 x 105) were injected s.c. into the shaved lateral flank. Tumor size was monitored on the indicated days. Tumor volume was calculated with the formula shown in Materials and Methods. Data are means ± SD of five (+/+) or seven (-/-) mice. *, p < 0.002 vs LT{alpha}+/+.

 
To determine whether LT{alpha}-/- mice can generate an effective defense against systemic tumor metastases, we next applied experimental metastasis models of syngeneic tumor cells and evaluated the formation of metastases in the knockout mice. LT{alpha}-/- mice exhibited significantly higher (p < 0.005) metastatic incidence in the form of experimental pulmonary metastasis following i.v. injection of B16F10; LT{alpha}+/+ mice exhibited 46 ± 28 (mean ± SD) foci per lung (n = 6), whereas LT{alpha}-/- mice exhibited 117 ± 29 foci per lung (n = 6). The mean number of melanoma colonies found in LT{alpha}-/- mice was 2- to 3-fold greater than that in the wild-type littermate controls. The lungs from LT{alpha}-/- mice were grossly enlarged by the metastatic disease, and the lung colonies formed in LT{alpha}-/- mice were markedly larger in size (Fig. 2Go). Additionally, most of the LT{alpha}-/- mice, but not wild-type mice, appeared to be severely cachectic on the day of the harvest. Multiple skin metastases and moderate to severe peritoneal dissemination were also observed in all knockout mice examined. Multiple liver metastases were found in five of six knockout mice and in two of six wild-type mice examined. Similar results were obtained for experimental metastasis of 3LL Lewis lung carcinoma cells (data not shown).



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  		FIGURE 2. Experimental pulmonary metastasis of B16F10 cells is enhanced in LT{alpha}-deficient mice. B16F10 cells (2 x 105) were injected i.v. from the lateral tail vein. Twenty-one days after injection, lungs were harvested and the number of surface metastatic colonies was counted under a dissecting microscope. Photograph shows three representative lungs from six wild-type (+/+) and six LT{alpha}-deficient (-/-) mice.

 
Splenocytes from LT{alpha}-/- mice exhibit lower NK cytotoxicity in vitro

Previous studies have demonstrated the involvement of NK cells in the inhibition of experimental and spontaneous metastases in several models (25, 26, 27, 28). Therefore, these data suggested a deficiency in NK activity in LT{alpha}-/- mice. We next performed experiments to evaluate the effect of the LT{alpha} deletion on in vitro NK cytotoxicity. Splenocytes were isolated from wild-type and LT{alpha}-/- mice, and cytotoxicity against NK-susceptible YAC-1 cells was evaluated. As shown in Fig. 3Go, unstimulated splenocytes from LT{alpha}-/- mice exhibited significantly less cytolytic activity against YAC-1 cells than did cells obtained from wild-type littermates (p < 0.02). Administration of polyICLC, a cytokine-inducing biologic response modifier, induces a rapid increase in the total number and the cytolytic activity of organ-associated and blood NK cells (21). In vivo stimulation with polyICLC markedly augmented the cytolytic activity of both wild-type and LT{alpha}-/- splenocytes. Splenocytes from wild-type mice stimulated by the i.p. injection of polyICLC displayed at 24 h a percentage specific cytotoxicity of 34.9% ± 2.5% (mean ± SE) against YAC-1 target cells at the E:T ratio of 100:1, whereas LT{alpha}-/- splenocytes that were stimulated similarly displayed cytotoxicity of only 20.4% ± 3.5% at the same E:T ratio (p < 0.04) (Fig. 3Go). Similar results were obtained for splenocytes stimulated in vitro with polyICLC or rIL-12 (data not shown).



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  		FIGURE 3. NK-mediated cytotoxicity of splenocytes is decreased in LT{alpha}-deficient mice. PolyICLC (10 µg) was injected i.p. into mice, and spleens were harvested 24 h after injection. Whole splenocytes were depleted of erythrocytes by hypotonic shock. Specific lysis of unstimulated and polyICLC-stimulated splenocytes against YAC-1 cells was evaluated by standard 4-h 51Cr release assay. Data are means ± SE of three independent experiments. *, p < 0.02 vs LT{alpha}+/+; **, p < 0.04 vs LT{alpha}+/+.

 
Spleens from LT{alpha}-/- mice contained 2- to 3-fold more leukocytes than that from wild-type littermates. Therefore, it was possible that the lower cytolytic activity observed in LT{alpha}-/- mice might be the result of a lower content of splenic NK cells. Indeed, flow cytometric analyses indicated that the percentage of NK1.1+CD3e- cells in spleens from wild-type mice was 2.6% ± 0.2% (mean ± SE, n = 6), whereas the NK level in LT{alpha}-/- mice was 1.2% ± 0.2% (n = 6; p < 0.001 vs LT{alpha}+/+). Twenty-hour culture in vitro in the presence of the stimulants did not significantly increase the number of NK cells in either type of mice.

Two distinct molecular pathways, perforin/granzyme-mediated and Fas-mediated pathways, are involved in tumor cell lysis by CTL and NK cells (29, 30, 31, 32). NK cell-mediated, perforin-dependent cytotoxicity is known to be crucial for the rejection of MHC class I-negative tumor cells (33), such as B16F10 melanoma. Sayers et al. (34) compared the contributions of these pathways in killing of the murine renal carcinoma cell line Renca and found that activated NK cells predominantly use the perforin/granzyme-dependent pathways and activated T cells use Fas/FasL-dependent pathways. To investigate the possible role of LT{alpha} in perforin regulation, immunoblot analysis was performed with partially purified splenic NK cells. Our data indicated that perforin protein was clearly detectable in NK cells from LT{alpha}-/- mice under both unstimulated and polyICLC-stimulated conditions, and the expression level was only slightly lower than that of the wild-type littermates (data not shown).

Overall, these data suggest that the reduced in vitro cytotoxicity of splenic NK cells from LT{alpha}-/- mice primarily resulted from a reduced frequency of these cells in the splenic leukocyte population, although contribution of a reduced cytotoxic potential of the individual NK cells cannot be excluded and should be further analyzed.

LT{alpha}-/- mice display a defective NK cell recruitment to parenchymal organs

The recruitment of leukocytes into sites of infection or tumor growth represents a critical early step in the development of an effective host defense. Therefore, we next analyzed NK cell recruitment to the parenchymal organs in response to exogenous stimuli. Lungs and livers from unstimulated LT{alpha}-/- mice contained an increased number of nonparenchymal cells (data not shown), consistent with the immunohistochemical observations reported previously (11). Fig. 4Go shows NK cell recruitment to the lung induced by polyICLC, an agent known to augment the organ-associated NK cell number (21, 35, 36). A dramatic expansion of the NK1.1+CD3e- population was observed in the wild-type mice 72 h after i.p. injection of polyICLC. The content of NK1.1+CD3e- cells was 6.5% ± 1.5% (mean ± SE) in unstimulated mice, and this elevated to 25.7% ± 0.9% after stimulation. Conversely, polyICLC failed to induce a marked NK cell increase in LT{alpha}-/- mice. The content of NK1.1+CD3e- cells was 1.3% ± 0.5% and 5.7% ± 3.2% in unstimulated and polyICLC stimulated mice, respectively. A significant difference was found between polyICLC-stimulated wild-type and LT{alpha}-/- mice (p = 0.026). In unstimulated conditions, the total number of NK cells per lung in LT{alpha}-/- mice was comparable with that in wild-type littermates (Table IGo) because of the increased leukocyte count in the LT{alpha}-/- mice. However, NK cell numbers in LT{alpha}-/- mice were significantly lower than in wild-type littermates after polyICLC stimulation (Table IGo). No appreciable change was found in NK1.1+CD3e+ cell populations after polyICLC stimulation in both strains (Fig. 4Go). Similar results were obtained for liver-associated NK cells (Table IGo and data not shown). These data indicate that LT{alpha}-/- mice show slower migration of NK cells to the parenchymal organs.



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  		FIGURE 4. NK cell recruitment to the lung is decreased in LT{alpha}-deficient mice. PolyICLC (10 µg) was injected i.p. into mice, and lungs were harvested 72 h after injection. Lung nonparenchymal cells were isolated by the procedures described in Materials and Methods, stained with PE-anti-NK1.1 and CyChrome-anti-CD3e mAbs (PharMingen), and subjected to flow cytometric analyses. Representative data from two independent experiments with groups of three mice are shown.

 

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  		Table I. PolyICLC-induced NK cell recruitment to the organs1

 
NK cell development from BM precursors is greatly suppressed in LT{alpha}-/- mice

It has been demonstrated that the rapid accumulation of NK cells in the liver induced by biologic response modifiers is mainly associated with the rapid NK cell development from BM precursors (35). Therefore, the impaired NK recruitment in response to exogenous stimuli may suggest a defective production of this cell population from BM precursors. To address the possible involvement of LT{alpha} in NK cell development, we cultured BM cells in the presence of IL-2 and/or IL-15 and estimated the efficiency of lytic NK cell development by cytotoxicity assay. In vitro stimulation with IL-2, IL-15, or the combination of these cytokines markedly augmented NK-mediated cytotoxicity in vitro in wild-type BM cells (Fig. 5Go, A and B), as reported previously (37). However, exogenous IL-2 and/or IL-15 could not efficiently induce the development of lytic NK cells in LT{alpha}-/- mice. Cytolytic activity of LT{alpha}-/- BM cells was inducible to some extent but was much lower than that of similarly stimulated wild-type BM cells (Fig. 5Go, A and B). These data suggested that production/maturation of lytic NK cells from BM precursors is impaired in LT{alpha}-/- mice, and this impaired production is likely due to a defective responsiveness to cytokine stimulation.



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  		FIGURE 5. Development of NK cells from cultured BM cells is impaired in LT{alpha}-deficient mice. BM cells were harvested from unstimulated mice and cultured for 4 days in the presence or absence of IL-2 and/or IL-15. Specific lysis of cultured BM cells against YAC-1 cells was evaluated by standard 4-h 51Cr release assay. A, Representative data from three independent experiments. B, Mean ± SE of three independent experiments (cultured with IL-2 + IL-15). *, p = 0.007 vs LT{alpha}+/+; **, p = 0.01 vs LT{alpha}+/+.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that LT{alpha}-/- mice have impaired antitumor surveillance defenses. In particular, syngeneic tumor cells were more metastatic and grew more rapidly in LT{alpha}-/- mice as compared with wild-type littermates. Additional analyses indicated that LT{alpha}-/- mice were defective in NK migration to parenchymal organs and NK cell development from BM cells induced in vitro. Our data are consistent with the previous reports demonstrating that mice with impaired NK activity manifest increased growth and metastasis of experimental tumors (25, 26, 27, 28).

Because LT{alpha}3 and LT{alpha}1ß2 can induce cell death through TNF receptors and LTßR (38, 39, 40), the effect of these molecules on the viability of inoculated tumor cells should be considered. Although we do not have data to demonstrate direct cytotoxic effects of LT molecules on B16F10 cells in vivo, the results in another tumor model argue against contribution of direct cytotoxicity. Indeed, despite the ability of Ab-LT fusion protein to induce apoptotic death of M24met melanoma cells in vitro, M24met growth could not be inhibited by systemic administration of this fusion protein to scid/beige mice (41), suggesting that LT molecules are not able to suppress the in vivo growth of tumor cells even if they are sensitive to LT-induced apoptosis. Therefore, we favor the hypothesis that LT contributes to the tumor rejection by stimulating the host immune response, not by killing the tumor cells directly.

LT{alpha}-/- mice completely lack peripheral lymph nodes; thus, it is possible that such an anatomical defect may affect the innate and/or adaptive antitumor defense of LT{alpha}-/- mice. However, previous studies demonstrated that BM is the major source of rapidly migrating NK cells in response to biologic response modifiers (35). Additionally, it was also shown that NK cell activation occurs systemically after i.v. injection of bacterial adjuvant or polyIC, and that the NK activity was augmented in spleens and peripheral blood more efficiently than in peripheral lymph nodes (42). These data suggest that peripheral lymph nodes may play a relatively minor role in NK recruitment and in systemic NK-mediated antitumor responses. We believe that the enhanced efficiency of experimental metastasis in LT{alpha}-/- mice is mainly due to the impaired production of lytic NK cells from BM precursors and recruitment to the organs and not to insufficient activation of NK cells resulting from the lack of peripheral lymph nodes.

Similarly, it is also conceivable that the impairment of some functions of tumor-specific CTL might contribute to the enhanced tumor growth and metastasis. Previous studies demonstrated that transfection of MHC class I and/or IFN-{gamma} gene to B16 cells (43) or injection of IFN-{gamma} retroviral vector to the B16 s.c. tumors (44) successfully induced a strong anti-B16 response mediated by CD8+ T cells. Additionally, in vitro treatment of B16 cells with IFN resulted in an augmented expression of MHC class I and an enhanced sensitivity to CTL (45, 46). However, inoculation of untreated parental B16 cells failed to induce detectable CTL activity (46). In fact, parental B16 melanoma cells are considered to be less immunogenic because of lower MHC class I expression on their cell surface (47). Additionally, it has been demonstrated that in vitro induction of allogeneic CTL response appears normal in LT{alpha}-/- mice (6). Therefore, it is unlikely that the enhanced tumor susceptibility in LT{alpha}-/- mice was largely due to defects in Ag-specific CTL responses.

The total number of NK cells per spleen in LT{alpha}-/- mice was similar to that of wild-type mice, but the NK cell content per organ was actually lower in the knockout mice, although the total number of leukocytes was increased. Because the NK1.1 cell surface molecule is expressed on C57BL/6 but not on 129/Sv NK cells (37), the detection of NK cells with anti-NK1.1 Ab may be complicated in C57BL/6-129/Sv mixed-background mice. To avoid this potential problem with LT{alpha}-/- mice initially generated on mixed C57BL/6-129/Sv background, we backcrossed these LT{alpha}-/- mice to the C57BL/6 background at least 10 times and always used wild-type control littermates obtained from LT{alpha}+/- heterozygous breeding pairs as our controls. Although our preliminary data suggest some down-regulated expression of perforin protein in LT{alpha}-/- splenic NK cells (data not shown), we concluded that the lower content of NK cells in LT{alpha}-/- spleens is the main reason for the suppressed in vitro cytotoxicity.

Importantly, our data showed that de novo production of NK cells from BM is impaired in the knockout mice. IL-2 and IL-15 failed to augment cytolytic NK cells in the in vitro culture of LT{alpha}-/- BM cells, and flow cytometric analyses also indicated that NK1.1+CD3e- cells were not efficiently produced from cultured LT{alpha}-/- BM cells stimulated in vitro with these cytokines (data not shown). PolyICLC is a strong inducer of several cytokines, including TNF and IFN-{gamma}, and augments NK cell development from BM precursors, migration to the organs, and cytolytic activity (21, 35, 36). Fogler et al. (23) showed that the VCAM-1/very late Ag-4 interaction is strongly enhanced by TNF rather than IFN-{gamma} after polyICLC treatment and is critical for the migration of newly recruited NK cells into parenchymal organs and tumor lesions. Okahara et al. (48) demonstrated that exogenous TNF up-regulates the expression of VCAM-1 on vascular endothelial cells and very late Ag-4 on B16 melanoma cells and enhances the experimental lung metastasis of B16. The exact regulatory role of LT{alpha} signaling in NK development remains to be determined. Although we cannot exclude the possibility that LT{alpha}, like TNF, plays a role in the regulation of endothelial adhesion molecules that are necessary for NK cell migration to parenchymal organs or tumor cell binding to the peripheral vasculature, the lower production of this cell population from BM precursors may significantly contribute to defective NK recruitment to the liver and enhanced tumor growth/metastasis observed in LT{alpha}-/- mice. The failure of exogenously added cytokines to induce NK cell development sufficiently in the culture of LT{alpha}-/- BM cells suggests an impairment in cytokine receptor pathways. In particular, LT{alpha} may control the expression of IL-2/IL-15Rß or the signal transduction pathway associated with this receptor system. Alternatively, the lack of LT{alpha} may result in defects in the biologic environment that is essential for NK activation and thus may indirectly affect the NK activities.

While this manuscript was in preparation, Smyth et al. (49) reported that TNF-deficient mice are defective in tumor rejection in the peritoneum. However, the functional characteristics of NK cells observed in the LT{alpha}-/- mice were quite different from those reported for TNF-/- mice. Contrary to our findings with the LT{alpha}-deficient mice, Smyth et al. (49) found that NK-mediated in vitro cytotoxicity was normal, but the NK recruitment to the peritoneum in response to i.p. injection of tumor cells was abrogated in the absence of endogenous TNF. In contrast, the NK response to polyIC, one of the components of polyICLC, was retained in TNF-/- mice. NK cell migration to the peritoneal cavity could be effectively stimulated by polyIC in vivo. In our experiments, polyICLC could induce only a small increase in the percentage of lung NK cells in LT{alpha}-/- mice. As suggested by Smyth et al. (49), TNF may make only a relatively minor contribution to the regulation of polyIC-stimulated NK cell migration to the peritoneum. At variance with these findings, our results suggest the importance of LT{alpha} in polyICLC-stimulated NK cell recruitment to parenchymal organs.

IFN-{gamma} has immunomodulatory effects on several cell populations, including NK cells. Previous reports demonstrated that excess tumor growth was observed in IFN-{gamma}-deficient mice (50). Thus, the impaired tumor control and NK activity observed in LT{alpha}-/- mice might be due to a down-regulated expression of IFN-{gamma}. However, studies using other biologic models suggested that IFN-{gamma} production is not impaired in mice with LT{alpha} deficiency (51, 52, 53). Therefore, it is unlikely that the defective NK activity in LT{alpha}-/- mice is the result of IFN-{gamma} down-regulation.

Although the specific molecular mechanisms of LT-mediated NK activation have not been investigated in detail, the overall evidence may suggest that signals coming through the LTßR are more important for NK cell function than those coming through TNF receptors p55 and p75. In fact, our preliminary data indicate that genetic inactivation of TNF receptor p55 did not affect the efficiency of experimental lung metastasis of B16F10 (data not shown). Considering the earlier report that surface LT species on human LAK cells can support their cytotoxicity by up-regulating effector-target adhesion (54), it is quite possible that the lack of the surface LT in LT{alpha}-/- mice may affect the NK cell adhesion required for NK recruitment and target cell lysis. To address such a possibility, blocking strategies both in vitro and in vivo with Abs or soluble ligands/receptors would be necessary. Alternatively, LT signaling may regulate the expression of other classes of adhesion molecules essential for interaction of NK cells with the endothelium, and such expression may be low in LT{alpha}-/- mice.

Overall, our findings in this study suggest that LT-LTßR signaling is important for development and recruitment of NK cells and, therefore, ultimately in NK-mediated host defense. The molecular mechanisms for TNF- and LT-mediated regulation of nonspecific cellular immune responses should be further addressed in future studies.


   Acknowledgments
 
We thank Drs. D. Kuprash, M. Watanabe, M. Lagarkova, I. Lyakhov, and K. Abe for assistance and helpful discussions and L. Drutskaya for genotyping mice. We especially thank Dr. Tomoaki Hoshino for technical support and instruction. We are indebted to Drs. S. Vogel, A. Anderson, J. J. Oppenheim, J. R. Ortaldo, and T. J. Sayers for critical reading of the manuscript.


   Footnotes
 
1 This project has been funded in whole or in part with U.S. federal funds from the National Cancer Institute, National Institutes of Health (contract NO1-CO-56000). S.A.N. is an International Research Scholar of the Howard Hughes Medical Institute. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Back

2 Address correspondence and reprint requests to Dr. Daisuke Ito, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, NCI-FCRDC, Building 560, Room 31-33, Frederick, MD 21702-1201. E-mail address: Back

3 Address correspondence and reprint requests to Dr. Sergei A. Nedospasov, Intramural Research Support Program, SAIC Frederick, NCI-FCRDC, Building 560, Room 31-70, Frederick, MD 21702-1201. E-mail address: Back

4 Abbreviations used in this paper: LT, lymphotoxin; BM, bone marrow; polyIC, polyinosinic-polycytidylic acid; polyICLC, polyIC and polyL-lysine stabilized in carboxymethyl cellulose. Back

Received for publication February 24, 1999. Accepted for publication June 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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