In this study, we demonstrate that Mϕ can recognize L5178Y tumor cells. Previous studies indicated that isolated ligation of CD40, NKG2D, and CD18 can be involved in activation of Mϕ (17
). Our current study dissects a functional hierarchy of these interactions in a polyvalent cross-talk between tumor cells and Mϕ. Even though CD154 is expressed in relatively low amounts on L1578Y cells (), its role in priming of Mϕ via CD40 was found to have a much greater stimulatory impact on Mϕ than Mϕ cross-linking via CD18 by ICAM-1 and ICAM-2, which are highly expressed on L5178Y cells. Similarly, whereas isolated blockage of NKG2D–H60 interactions had no noticeable effect on Mϕ in vitro
secretory and anti-tumor activities, it was required, in addition to blockage of CD40–CD154 and CD18–ICAM-1/-2, for abrogation of Mϕ activation by L5178Y cells. Thus, the Mϕ-tumor cell cross-talk that ultimately leads to induction of anti-tumor Mϕ involves concurrent engagement of multiple receptor-ligand pairs.
It also appears that tumor cells should simultaneously express several stimulatory ligands in order to induce Mϕ activation. In this regard, normal syngeneic mouse splenocytes (essentially comprised of CD3+
T cells [~35%], B220+
B cells [~40%], CD11b+
mononuclear cells [~10%], and CD49b+
NK cells [5–7%]) did not prime Mϕ, in contrast to priming by L5178Y cells. Whereas most of the splenocytes expressed ICAM-1 and ICAM-2 (in a very heterogeneous fashion), only a small fraction (mostly CD4+
T cells, less than 15% of total naïve splenocytes) expressed CD 154 (and only at very low levels), and no splenocytes expressed ligands of NKG2D (data not shown). Activation of splenocytes with LPS and CpG to produce IL12 and IFN-γ (24
) might facilitate Mϕ activation and induce moderate levels of anti-tumor effects in vitro
without involvement of the CD40/NKG2D/CD18-dependent mechanism; however, in our in vitro
experiments, splenocytes did not trigger NO production by Mϕ. Indeed, culturing Mϕ with 1×104
L5178Y cells led to only marginal activation of Mϕ (, ), whereas culturing Mϕ with 2.5–5×104
L5178Y cells led to robust activation of Mϕ.
Several reports have previously demonstrated that the Mycoplasma arginini
– infected L5178Y and YAC-1 cell lines could activate thioglycollate-elicited peritoneal Mϕ in the presence of LPS or exogenous IFN-γ to mediate in vitro
anti-tumor effects via NO- and TNF-α-dependent mechanisms (28
). Results of those studies differed in the sensitivity of the model to the suppressive effects of PG-E2, but both agreed that the priming effects could be transferred by the tumor cell-conditioned supernatants or mediated by paraformaldehyde-fixed tumor cells. In our study, we tested L5178Y cell line for Mycoplasma spp.
contamination, and found no evidence for infection. The absence of Mycoplasma spp.
in L5178Y cells was also confirmed at a DNA level by using fluorescent probes (data not shown). Most importantly, our results, unlike those published by Young et al
), and Ribeiro-Dias et al
), suggest that priming of Mϕ required direct cell contact with viable L5178Y cells; Mϕ could not be primed by either L5178Y cell-conditioned supernatants () or fixed L5178Y cells. Furthermore, separation of Mϕ and viable L5178Y cells by the transwell also abrogated priming of Mϕ. Whereas we cannot exclude a contribution of soluble, LPS-induced tumor cell-derived factors, such as IL-1 (30
), our results suggest that membrane-bound, but not soluble factors are important for induction of cytotoxic Mϕ in our in vitro
model. The inability of fixed L5178Y cells to prime Mϕ might also be due to potential toxic effects of fixed L5178Y cells on Mϕ (such as leaching of paraformaldehyde). However, our preliminary data argue against this, because co-culture of fixed L5178Y cells with viable L5178Y cells did not affect proliferation of viable L5178Y cells as measured by 3
H-TdR incorporation (data not shown). Complete absence of Mϕ priming by fixed L5178Y cells in our in vitro
experiments could be explained by denaturation of CD154 subunits by paraformaldehyde, or by inability of fixed L5178Y cells to upregulate CD154 expression upon contact with Mϕ in the presence with LPS, which might be important for compensation of CD40-downregulation on L5178Y-primed Mϕ. Lastly, loss of L5178Y cell membrane fluidity and inability of PF-L5178Y cells to mobilize CD154-, H60- or ICAM-1- and ICAM-2-containing membrane rafts to the site of direct contact with Mϕ might also lead to this loss of Mϕ-priming properties, provided that the cells express no other priming substances such as those associated with Mycoplasma spp.
Despite high concentrations of NO produced by L5178Y cell + LPS-stimulated Mϕ, L5178Y cell proliferation in the TW-chamber was not suppressed when co-cultured with L5178Y cell + LPS-stimulated Mϕ in the bottom of the TW-chamber system. In contrast, direct contact with Mϕ resulted in substantial anti-tumor effects, suggesting membrane-mediated death signaling. In this regard, we found only very limited expression of membrane-bound TNF-α, FasL, and TRAIL on the surface of Mϕ after their exposure to L5178Y cells and LPS (data not shown). The potential roles of other factors in L5178Y + LPS-stimulated Mϕ-mediated cytotoxicity remain to be clarified.
Flow cytometric analysis of the immunophenotype of Mϕ and L5178Y cells revealed that exposure of these cells to each other results in changes in expression of some of the antigens forming certain receptor-ligand pairs. Hence, a relative decrease (7–25%) of CD40 expression on Mϕ coincided with upregulation (31–88%) of CD154 on L5178Y cells, after these cells were cultured in the presence of LPS. Similarly, upregulation (44–127%) of NKG2D on Mϕ coincided with a slight decrease (11–16%) in expression of H60 on L5178Y cells. The observed alterations of the CD40–CD154 pair expression could favor recognition of L5178Y cells via CD154. The decreased expression of H60 on L5178Y cells following contact with Mϕ is in agreement with the observation by Bui et al. (31
) that type I IFN down-regulated H60 in some tumors, and could be associated with mechanisms of immune evasion (32
); yet we observed upregulation of NKG2D expression on L5178Y lymphoma-primed Mϕ in the presence of LPS (). Unlike the increased MHC-I we have noted on tumor cells escaping from NK cell effects (33
), there was no substantive change in MHC-I expression on L5178Y cells cultured with Mϕ (). Treatment of L5178Y cells with αMHC-I mAb did not alter secretory and anti-proliferative functions of Mϕ (). In agreement with this, we have recently demonstrated that αCD40-induced Mϕ-mediated in vivo
anti-tumor effects can be effectively induced against both highly-and weakly-immunogenic tumors, independent of MHC-I expression (34
Although the expression of NKG2D and its potential role in Mϕ activation and Mϕ–mediated killing has been controversial (17
), our data document upregulation of cell surface NKG2D on Mϕ when cultured together with L5178Y cells and LPS. Furthermore, we demonstrate the abrogation of Mϕ activation in these cultures when Mϕ–based CD40, CD18, and NKG2D molecules are simultaneously prevented from recognizing their respective ligands. These data are consistent with the demonstration that mouse Mϕ activated by LPS and IFN-γ upregulate NKG2D and these activated Mϕ kill tumor cells that express adenovirus E1A in an NKG2D-dependent manner (J. Routes, Medical College of WI, manuscript in preparation, personal communication).
In a variety of immunological reactions, Mϕ can serve as antigen-presenting cells providing stimuli for T cells by MHC class II-associated exogenous antigens as well as via a number of co-stimulatory molecules. However, engagement of certain cell surface molecules on Mϕ causes reciprocal signaling, resulting in Mϕ activation. Several Mϕ membrane-associated surface molecules, including CD40, MHC class II, and CD18, could be involved in this reciprocal stimulation associated with modulation of signaling pathways, production of cytokines, and changes of immunophenotype. Frequently, ligation of more than one type of molecule on the surface of Mϕ is required for induction of cytokines. For example, it was shown that interaction of Mϕ with T cells via CD40–CD154 leads to accumulation of IL-12p40 in Mϕ; at the same time, additional contacts via the MHC class II/TCR pair was needed to trigger Mϕ to produce IL-12p35, thus enabling production of functional IL-12p70 (36
). Our results demonstrate that L5178Y lymphoma cells express high amounts of H60 as well as ICAM-1 and ICAM-2. Thus, it is possible that H60 and ICAM-1/-2 molecules could synergize with CD154 in Mϕ activation. The potential mechanisms of these additional interactions between Mϕ and L5178Y cells remain to be clarified, but could range from a simple anchoring of the ICAM+
-tumor cells to the surface of CD18+
) (to facilitate cross-talk via CD40-CD154 and NKG2D-H60 pairs) to an active recruitment of a Toll/IL-1 receptor family-like cascade to modulate TLR-signaling (39
). The latter effect on adapter proteins of TLR-signaling pathways might partially explain selective sensitivity of L5178Y-primed Mϕ to LPS but not CpG, although the most likely explanation is that peritoneal mouse Mϕ readily express TLR4 (40
), whereas TLR9 expression is negligible without proper stimulation (21
Alternatively, the ability to prime Mϕ may be restricted to the cells of a certain embryonic origin. Thus, L5178Y cells and A20 cells of lymphoid origin could prime Mϕ [for A20 cells, presence of the exogenous αCD40 () or other CD40L-expressing cells () was required], whereas B16 melanoma cells (neural crest origin) and mouse fibroblast L cells (mesenchymal origin) did not have this ability even in the presence of exogenous αCD40 (B16, ) or after transfection with functional CD40L (CD40L-L, ). L5178Y and A20 cells have also been able to facilitate priming by other CD154+ cells (CD40L-L), but this co-stimulatory ability has been lost when cells were paraformaldehyde-fixed (not shown). Importantly, this effect was independent from the CD154-expressing status of these lymphoma cells, as A20 cells are CD154-negative (). Hence, it remains possible that some soluble factors or other surface co-stimulatory molecules expressed by lymphoid cells in the process of initial cross-talk between tumor cells and Mϕ, are involved in the process of Mϕ priming.
The conclusions made from in vitro experiments were consistent with data obtained in our in vivo tumor model. Intraperitoneal implantation of L5178Y cells resulted in alterations of phenotype of resident Mϕ (upregulation of MHC-II and downregulation of CD80 and CD86), as well as expression of IFN-γ and TNF-α, but not IL4 or IL10 (). It is unclear whether these immunological changes are solely due to interaction of peritoneal Mϕ with L5178Y cells, or if this is a result of the cross-talk between Mϕ and tumor cells in the presence of very low, “physiological” concentrations of circulating endotoxin. However, when small doses of LPS were given to the tumor cell recipients, Mϕ expressed much higher levels of MHC-II, CD80, CD86, IFN-γ, and TNF-α than after the treatment with L5178Y cells or LPS alone. Surprisingly, there was a significant increase of production of IL10, which is known to play an important role in regulation of Mϕ activation. Discrepancy in magnitude of Mϕ phenotypic changes seen in in vitro vs. in vivo models could be explained by overall differences in experimental conditions. Thus, in in vitro experiments () Mϕ and L5178Y cells were in constant contact for the entire duration of the experiment, and the volume of distribution of Mϕ-derived and potentially L5178Y cell-derived soluble factors was confined to 0.2 ml. On the other hand, in the in vivo experiment interactions between Mϕ and L5178Y cells could be less durable and the volume of distribution of soluble factors was much larger and potentially affected by physiologic metabolic processes. Even if the overall phenotypic changes are less pronounced in vivo, they could be considered as proof of principle of active Mϕ-L5178Y cell cross-talk resulting in phenotypic and functional alterations of Mϕ.
Efficient tumor recognition at the early stage of tumor progression might be a critical component of cancer immune surveillance. The state of “dormant” cancer exemplifies immune-mediated tumor growth restriction in the absence of active exogenous immunostimulatory interventions. L5178Y lymphoma in DBA/2 mice has been used for decades to study this biological phenomenon (41
). Both cytotoxic T cells and Mϕ are involved in maintaining L5178Y lymphoma in the dormant non-progressive stage of disease (42
). In the present study, we suggest an additional mechanism of this immunological lymphoma surveillance: L5178Y cell-induced priming of Mϕ to endotoxin, which is naturally present in biological fluids in minute concentrations (45
), might be involved in Mϕ activation and result in anti-lymphoma effects in vivo
. A number of reports demonstrate that human T cell lymphomas, as well as other cancers, can express CD154 (46
). Whether human Mϕ can recognize autologous cancer cells via similar mechanisms, shown here for L5178Y cells, remains to be determined.