We have demonstrated that TIM-2 is expressed in localized areas of the spleen, liver, and kidneys, and that it binds H-ferritin, but not L-ferritin. Further, after binding of H-ferritin to TIM-2 on the cell surface, H-ferritin is internalized into endosomes.
In the spleen, we found that TIM-2 is expressed on all B cells, with expression at higher levels on GC B cells. Thus, different members of the TIM family are expressed in different subsets of immune cells; TIM-2 is on B cells, whereas TIM-1 is inducible on Th2 cells, and TIM-3 is inducible on Th1 cells. TIM-4 initially was identified on splenic stromal cells, but recent studies indicated that it also is expressed on antigen-presenting cells, where it serves as a ligand for TIM-1 (7
). We have not detected TIM-2 on resting T cells, nor have we been able to induce its expression on T cells in vitro by treatment with mitogen (ConA) or by stimulation of T cells under conditions that induce Th1 or Th2 cells (unpublished data). However, the induction of TIM-1 or -3 on T cells requires repeated stimulation, and we may not have identified proper conditions to induce the expression of TIM-2 on T cells. Regardless, although TIM-2 is 66% homologous to TIM-1, these two TIM receptors differ in their expression on lymphocytes.
Our demonstration that TIM-2 binds to H-ferritin is the first identification of a cell surface receptor for ferritin, although the existence of ferritin receptors was postulated previously, based on saturable binding of ferritin to the surface of several cell types, including B and T lymphocytes. Thus, binding of H-ferritin to lymphocytes is increased in mitogen-stimulated cells, and purified H-ferritin forms surface patches on T cells, after which it is endocytosed into lysosomes (23
). Studies using MOLT-4 human T cells indicated that they express a receptor for H-ferritin with an association constant of ~7 × 10−7
L/mol, and that the receptor is increased on proliferating cells (25
). Biochemical evidence for an H-ferritin receptor also was found on liver cells, including activated liver lipocytes (33
). In addition, specific ferritin binding was demonstrated on erythroid precursor cells (35
), brain tissue (37
), and placental membranes (39
In one report, a putative ferritin receptor was purified from human liver (40
). Although the molecular weight of this receptor, 53 kD, is generally consistent with the predicted size of glycosylated TIM receptors, it was not identified further, and the specificity of this receptor for H-ferritin was not shown. The liver seems to express at least two binding sites for H-ferritin; one binds L- and H-ferritin with equal affinity, whereas the second, like TIM-2, binds only to H-ferritin (33
). In healthy individuals, circulating ferritin is predominantly L-ferritin. It was suggested that the first receptor, which binds H- and L-ferritin, may serve to regulate levels of serum ferritin, whereas the H-ferritin receptor may subserve independent cellular functions in response to a selective increase in H-ferritin.
Ferritin is increased in inflammation, and we showed previously that TNF-α and IL-1 activate transcription of the H-ferritin gene in an additive manner, providing at least one mechanism by which H-ferritin may be increased in inflammation (42
). The sources of circulating ferritin are not defined; however, our cloning of H-ferritin from a macrophage line is in accord with previous studies of H-ferritin production by macrophages, including studies that demonstrated that LPS stimulates H-ferritin production by J774 and RAW264.7 macrophage cells, both of which produced an LPS-inducible ligand for TIM-2 (44
). Recent studies have shown that transcription of H-ferritin is induced by NF-κB, and the consequent interaction of H-ferritin with iron serves to buffer the generation of reactive oxygen species, an activity that is required for the capacity of NF-κB to counter TNF-α–induced apoptosis (19
). Our studies demonstrate that TIM-2 permits the endocytosis of H-ferritin, revealing that cellular levels of H-ferritin are not dependent solely on transcription. This finding opens a new pathway into understanding the role of ferritin in cell function.
The properties of TIM-2 that are required for endocytosis of H-ferritin by TIM-2 remain to be elucidated. The cytoplasmic domain of TIM-2 has three tyrosine residues. The membrane-proximal tyrosine is part of a YxxM motif, which has been associated with endocytic localization as well as with activation of PI3 kinase (46
); however, the proximity of this motif to the inner leaflet of the cell membrane may render it nonfunctional. The other two tyrosines are not part of known signaling or targeting motifs, but one motif (E-[ED]-x-x-Y-x-x-E) is conserved through several TIM receptors, which suggests functional significance.
By IHC of the liver, mouse TIM-2 is expressed highly in bile ducts and, to a lesser extent, in hepatocytes. In accord with this, we find high levels of TIM-2 transcripts in the liver (unpublished data). The expression of TIM-2 in bile duct epithelial cells suggests that it may be involved in the transport of ferritin into or out of bile. Ferritin excretion into bile is believed to involve lysosomal exocytosis, but the mechanisms of excretion are not well defined (47
). From our studies, it is possible that this process involves TIM-2. Humans express high levels of TIM-1 in the liver; however, in the mouse, levels of transcripts for TIM-2 expression are 100–1,000 times higher than levels of transcripts for TIM-1 (unpublished data). Although the localization of TIM-1 in the human liver has not been described, these results suggest the possibility that, in the liver, mouse TIM-2 may be a functional ortholog of human TIM-1.
No human ortholog for mouse TIM-2 has been identified, although mouse TIM-2 is only slightly less homologous to human TIM-1 than is mouse TIM-1. Thus, human TIM-1 may share functions with mouse TIM-2 and -1, including the capacity to bind H-ferritin. Alternatively, the capacity of mouse TIM-2 to bind H-ferritin may have been usurped by a different human receptor. Whatever the nature of the human ferritin receptor, the expression of TIM-2 in mice roughly parallels that of known ferritin binding sites in humans. Thus, the expression of TIM-2 on mouse lymphocytes and hepatic cells corresponds with binding of H-ferritin to human lymphocytes and liver cells, except that we have not identified TIM-2 on resting T cells. However, TIM-2 is expressed on mouse EL-4 thymoma cells (unpublished data), and there may be conditions under which its expression is induced on fresh T cells. Studies are in progress to define the role of human TIMs in ferritin binding.
Our studies also may bear on the role of H-ferritin in malignancy. H-ferritin is increased often in malignancy, and its expression correlates with poor prognosis. Thus, the expression of transcripts for H-ferritin by breast cancer cells is an adverse prognostic indicator (49
). Similarly, in ovarian cancer, metastatic cells express higher transcripts for H-ferritin than do nonmetastatic cells (50
). In melanoma, levels of circulating H-ferritin are elevated, and the levels of H-ferritin correlate with levels of CD4+
regulatory T cells (51
). Further, H-ferritin was shown to activate regulatory T cells through mechanisms that require dendritic cells (52
). Studies in rats similarly showed an up-regulation of H-ferritin during the induction of hepatocellular carcinoma (53
). Our studies identify a receptor in mice through which H-ferritin may selectively alter cell functions, including immune functions. It will be of interest to pursue the possibility that the production of H-ferritin by malignant cells alters the host response through the TIM-2 ferritin receptor.
As an additional note, while this paper was under review, Chakravarti, et al. demonstrated that transcripts for TIM-2 can be induced selectively in Th2 T cells (54
). We did not detect elevated levels of TIM-2 transcripts in freshly isolated splenic T cells. In preliminary results we did, like Chakravarti, et al., find transcripts for TIM-2 in T cells after activation in vitro, but as noted in Results we have not defined conditions that will induce detectable levels of TIM-2 on the surface of T cells. We are testing the possibility that this may be regulated by H-ferritin.