Epithelial cells are important in initiating an innate immune response against external pathogens in tissues such as lung, intestine and mammary gland . Receptors to external pathogens, NFκB activation , and cytokine secretion together with production of reactive oxygen and nitrogen species production are critical for local elimination of the pathogen independent of immune cells as well as for activation and recruitment of immune cells to the site of infection .
This study investigated the co-regulation of IL-6 secretion and NO production by ET in SCp2 mouse mammary secretory epithelial cell cultures, with prediction of their co-induction by ET as known for immune cells . However, contrary to expectation, they showed different temporal patterns of response to ET in SCp2 mouse mammary secretory epithelial cells. The delayed response of NO relative to IL-6 was observed for both the secreted proteins and their mRNA expression (Figure 1). Although the observed delay in NO production might be partly explained by the time required for iNOS to synthesize NO and the rate of the conversion of the latter to a more stable nitrite (NO2
-) form, the observed delay in expression of iNOS mRNA which was not induced before 3 h post-ET (Figure 1D) compared to that of IL-6 mRNA induced at 1 h post-ET (Figure 1C), indicates differences in the regulation of expression of IL-6 and iNOS as ET-induced respondents.
Treatment of SCp2 cells with different doses of exogenous mouse recombinant IL-6 protein at doses (0.1, 1, and 10 nM) representing respectively 1/10, 1, and 10 × the levels of secreted IL-6 protein found in the medium of ET-treated SCp2 cells, did not induce expression of iNOS when applied alone in the absence of ET (data not shown), suggesting that the expression of iNOS mRNA was induced in response to ET alone and not in response to IL-6, and thus the delay in iNOS expression is due to a different control mechanism than that of IL-6.
We also investigated the effect of ET on the binding activity of p65 and p50, the major NFκB subunit types known to be involved in ET signaling via the rapid MyD88-dependent pathway (reviewed in ). The results in Figure 4 showed a rapid activation of both p65 and p50 at 1 h post-ET; however, while p65 activation was transient, activation of p50 was sustained through 6 h post-ET. Although others described the activation of p65/p50 NFκB via the rapid MyD88-dependent pathway involving IKK-dependent IκB phosphorylation and ubiquitin-dependent degradation (reviewed in ), other reports suggested that the regulation of p50 might involve additional pathways other than IκB degradation . In any case, the difference in p65 and p50 activation suggests that the difference between ET-induced IL-6 and iNOS mRNA expression might involve different pathways of NFκB activation; one that is IκB degradation-dependent while the other is not; therefore, inhibition of the IKK complex that phosphorylates IκB and labels it for degradation would modulate only the IκB dependent NFκB activation pathway and its target genes.
Consistent with the difference in regulation of both IL-6 and iNOS mRNA expression, Wedelolactone treatment inhibited only ET-induced IL-6 mRNA expression but not that of iNOS. These results suggest that while IL-6 mRNA expression is likely dependent on the rapid MyD88-dependent NFκB activation pathway, iNOS mRNA expression likely involves the delayed MyD88-independent NFκB activation pathway. Kawai et al (2000) showed that ET-induced IL-6 production was impaired in MyD88-/- mouse macrophages , while Schilling et al (2002) reported that expression of iNOS but not that of IL-6 mRNA was enhanced by ET-induced IFNγ in mouse macrophages . In our study, IFNγ was not significantly induced in SCp2 cells treated with ET (Figure 9). Hence, these studies, together with our results in epithelial cells suggest that the differential regulation of ET-induced IL-6 and iNOS by NFκB involves different kinetics and alternate pathways of NFκB activation for each whereby IL-6 mRNA expression is likely induced by the rapid MyD88-dependent activation pathway of NFκB but iNOS mRNA expression might involve the slower MyD88-independent activation pathway of NFκB. Furthermore, the observed delay in expression of iNOS compared to that of IL-6 mRNA coincides with reports describing a role for IL-6 in acute inflammation and in the transition to chronic inflammation , in contrast to NO that was proposed to limit inflammation .
Though iNOS is the induced form of NOS upregulated in response to ET in the SCp2 secretory mammary cells used in this study, other studies described a possibility for induction of mRNA expression of supposedly constitutive eNOS and nNOS or the regulation of eNOS by phosphorylation . Others also showed that the constitutive activity of eNOS can be modulated by phosphorylation of amino acid residues serine 1177 (Ser 1177) and/or threonine 495 (Thr 495) in rat heart and endothelial cells . In this study, we were unable to show conclusive qPCR amplification or modulation of protein expression of either eNOS or nNOS in response to ET (data not shown). Thus, our data suggest that ET-induced NO in SCp2 mammary epithelial cells is due mainly to iNOS expression and activity.
Moreover, by global immunodot-blot assay for ET-induced cytokines, we found that SCp2 mammary secretory epithelial cells are capable of producing inflammatory cytokines and chemokines similar to immune cells. The cytokines that were significantly upregulated in response to ET from immunoblot analysis were GCSF and IL-6 (Figure 9). GCSF is involved in monocyte chemotaxis as well as cell differentiation, suggesting that mammary secretory epithelial cells, likely to be the first cells in contact with infecting bacteria of the mammary gland, play an important role in innate immunity and in recruiting immune cells and orchestrating their function. IFNγ was present but was not significantly induced in ET-treated SCp2 cells.
The present study also demonstrates that ET-induced IL-6 secretion and NO production are differentially regulated by the microenvironment of SCp2 cells in culture. Upon induction of cell differentiation either by addition of EHS or by SCp2:SCg6 co-culture, we investigated the role of EHS and SCp2-SCg6 interaction on IL-6 secretion and NO production in SCp2 cells alone or in SCg6 co-cultures with or without ET-induced inflammation. While EHS had no effect on ET-induced IL-6 secretion (Figure 6D), SCp2 cells cultured in the presence of EHS showed a slight but significant increase (~25%) in NO production in response to ET compared to SCp2 cells on plastic (Figure 6E). Despite differential regulation of the secreted products, ET-induced iNOS mRNA had similar temporal patterns of expression regardless of substratum (data not shown). Whether the observed increase in NO in the medium was due in part to increased chemical stability of NO in the presence of EHS was not investigated. Similarly, IL-6 mRNA expression patterns in response to ET were not affected by EHS (data not shown).
However, there was a strong and unexpected spontaneous induction of IL-6 when SCp2 cells were cultured on a monolayer of SCg6 cells; IL-6 concentrations increased dramatically to several-fold higher than for either SCp2 or SCg6 cells alone, independent of ET treatment and cell number (Figure 7A &7B). The concentration of IL-6 in control co-culture cells was higher than the sum of IL-6 secreted in both control SCp2 and SCg6 cells cultured independently, showing a strong synergistic effect of SCg6-SCp2 interaction on IL-6 secretion even in the absence of ET (Figure 7A &7B). These results are interesting in light of reports in the literature of a role for IL-6 in cell-cell association . In marked contrast, growth of SCp2 cells on an SCg6 monolayer seemed to reduce ET-induced NO response although NO basal levels were not affected (Figure 7C), confirming that the previously shown differential regulation of IL-6 and NO extends to SCp2 cells in co-culture. Despite the intriguing effect of SCp2:SCg6 co-culture on spontaneous and induced inflammation, any inference as to how the effects of SCp2:SCg6 interaction would relate to the mammary gland in vivo requires caution since SCg6 cells were described as a malignant mammary cell line having lost their responsiveness to ECM regulation and were shown to induce tumors when injected into athymic nude mice . Whether SCg6 cells result in the tumor or induce transformation in epithelial cells is not yet clear; however, the induction of a spontaneous inflammatory response in SCp2 cocultured with SCg6 may be related to the predicate of chronic inflammation leading to cancer in epithelial tissues . Our findings become even more interesting in light of studies by Talhouk et al (2008) and Gudjonsson et al (2002). The first study underlines the importance of heterocellular interactions between SCp2 and SCg6 cells in enhancing expression of connexins and of catenin cell protein interactions as expected for more complete SCp2 cell and epithelial differentiation in vitro , while the second emphasizes the importance of myoepithelial cells in maintaining epithelial cell polarity and the bilayered structure of epithelial cells and myoepithelial cells in the normal mammary tissue .
The ratio of secretory epithelial to myoepithelial cells varies in ductular vs. alveolar mammary epithelia , with the ratio of secretory epithelial to myoepithelial cells low in duct tissue and higher (>1) in the alveolus with dominance therein by the monolayer of secretory epithelial cells surrounded by a discontinuous layer of myoepithelial cells . Therefore, we varied the ratio of SCp2 to SCg6 cells in the co-culture by plating SCp2 cells on a monolayer of SCg6 cells at SCp2:SCg6 cell plating ratios of 1:4, 1:2, 1:1, and 2:1. SCp2:SCg6 co-culture induced a dramatic 5 to 9-fold increase in basal levels of IL-6 secretion in control non-ET treated cells compared to that in SCp2 or SCg6 cells alone on plastic (Figure 8A vs. 8B). The net increase of IL-6 secretion from ET-induction was independent of cell number in SCp2:SCg6 co-cultures, indicating that cell-cell interaction affected only basal secretion of IL-6 proteins but not that induced by ET (Figure 8B). In contrast to IL-6, while basal NO levels varied little in SCp2 cells on plastic (Figure 8C), ET-induced NO production increased significantly (by 15 to 20-fold) only at high SCp2 cell plating density (4 and 8 × 104 cell/cm2) (Figure 8D) compared to basal non-ET treated levels; emphasizing again the differential regulation of IL-6 and NO in mammary epithelial cells. It remains intriguing but unknown whether or how the cellular ratios and influence thereof on regulation of the inflammatory responses in mammary epithelial cells as shown here might relate to the in vivo ratios and associations of secretory epithelial to myoepithelial cells within the quiescent adult human mammary gland and its elevated risk of developing breast cancer [35, 36].