In this study we have shown that TIM-4 and TIM-1 recognize PS on the surface of apoptotic cells. Transfection of TIM-4 or TIM-1 into NIH-3T3 cells greatly augmented their capacity to phagocytose apoptotic cells. This phagocytic activity was completely blocked by liposomes containing PS but not PC, PI, or PE. We identified mAbs against mouse and human TIM-4 and TIM-1 that would block binding to PS and phagocytosis. All the blocking mAbs mapped to the IgV domain on which the PS binding cavity is located. Consistent with a role in recognition of apoptotic cells, TIM-4 is highly expressed on phagocytic cells such as peritoneal macrophages, tingible-body macrophages, and dendritic cells. Tingible-body macrophages are the cells that engulf apoptotic cells in lymphoid tissue (Smith et al., 1991
PS comprises about 7% of the phospholipid of a cell but is compartmentalized to the inner leaflet of the cell membrane. The redistribution of PS to the external surface of the plasma membrane is a key element of apoptotic cell recognition and is a molecular cue that dying cells should be phagocytosed (Fadok et al., 1992
; Verhoven et al., 1995
). For example, healthy Jurkat cells expose about 0.9 attomol (600,000 molecules) per cell of PS which rises to about 240 attomol/cell late in apoptosis (Borisenko et al., 2003
). Macrophages inefficiently phagocytose cells expressing this low, healthy level of PS and phagocytosis increases only after a threshold level of about 5 attomol/cell is passed and plateaus above 25 attomol/cell (Borisenko et al., 2003
Several receptor combinations have been identified that participate in the recognition of PS on the cell-surface of apoptotic cells including Milk fat globule EGF-factor 8 (MFG-E8) (Hanayama et al., 2002
) and Growth arrest specific gene 6 (GAS6) (Scott et al., 2001
). These are two component systems composed of a soluble PS “tethering” molecule such as MFG-E8 or GAS6 and a membrane bound “tickle” molecule such as αV
integrin or Mer responsible for recognition of the “tether” and internalization and signaling. A PS receptor on the surface of macrophages has been described; however, the candidate molecule cloned by phage display (Fadok et al., 2000
) has been discredited (Williamson and Schlegel, 2004
). TIM-4 is a likely candidate for this macrophage PS receptor with the IgV domain comprising the “tether” that binds PS and the extended mucin domain providing a “tickle” to the TIM transmembrane and cytoplasmic domains. Recognition of PS by TIM-4 and TIM-1 was highly specific whereas other PS binding molecules such as MFG-E8 (Hanayama et al., 2002
), GAS6 (Nagata et al., 1996
; Nakano et al., 1997
), β2-glycoprotein (Balasubramanian et al., 1997
) and lectin-like oxidized low-density lipoprotein receptor 1 (Oka et al., 1998
) bind not only PS but other anionic phospholipids.
Phagocytosis of apoptotic cells is evolutionarily ancient and PS-recognition molecules such as MFG-E8 and GAS6 are broadly expressed in tissues. In contrast, expression of TIM-4 is highly restricted to professional APC and testis, suggesting an immunologically restricted function in recognition of apoptotic cells. Given the redundant systems that recognize apoptotic cells, the strong effects of TIM-1 or 4 mabs or fusion proteins are impressive.
The binding of TIM-1 or TIM-4-Ig to apoptotic cells was not species-restricted and apoptosis could be induced by methods including Fas-mediated, etoposide, cytokine deprivation, and starvation of cells, or Ca/ionomycin induction of eryptosis in RBC. Using strips coated with a variety of phospholipids, we showed that TIM-4 and TIM-1 bound to PS and not other phospholipids. All healthy long-term cell lines showed a low level of binding of TIM-1 and TIM-4, consistent with their low but detectable level of exposure of cell surface PS as measured by anti-PS mAb or AnnexinV-FITC (data not shown). In contrast, TIM-1 and TIM-4 did not bind to T cells from freshly isolated blood (data not shown). This is consistent with the fact that in vivo, cells exposing PS are rapidly phagocytosed whereas in vitro cell lines are not monitored as stringently since professional phagocytic cells are not present.
The co-crystal structure of PS bound to TIM-4 (Santiago et al., submitted) shows a direct interaction not mediated by intervening molecules. The specific binding of PS to TIM-4 is due to the unique structure of TIM IgV domains. The four non-canonical cysteines found in all TIM family members form disulfide bonds that reverse and fix the folded conformation of the long CC′ loop onto the GFC β sheet (Cao et al., 2007
; Santiago et al., 2007
). Instead of the relatively flat binding surface found in most Ig superfamily ligand recognition surfaces, the CC′FG loops form a narrow cavity with dimensions of approximately 7 × 9 × 11 angstroms. The outer lips of the cavity are composed of hydrophobic amino acids positioned to interact with the hydrophobic fatty acid region of PS. The bottom of the cavity is composed of charged amino acids that capture a metal ion and coordinate with the charged head of the PS. The recognition of PS by macrophages has been shown to be stereospecific for the L-isomer of serine and the D-isomer is not recognized (Hoffmann et al., 2005
). The co-crystal structure of the L-isomer of PS with TIM-4 shows this stereospecificity and the D-isomer would reverse the charge interactions within the binding pocket and be repulsed because of the juxtaposition of positive to positive and negative to negative.
Mutation of any of the amino acids (119–122, WFND) that line this binding cavity eliminates binding to PS and phagocytosis. The 9F4 mAb that blocks TIM-4 binding to PS maps to this epitope. The structures of mouse and human TIM-1 and TIM-4 are very highly conserved in this region whereas TIM-2 is completely different (--AF where -indicates a gap in the aligned sequences). This likely explains the similar recognition of PS by TIM-4 and TIM-1 and the different ligand binding of TIM-2. Clearly, TIM-2 is an outlier in the TIM family and binding to proteins including Semaphorin4A and h-Ferritin has been reported. TIM-1 and TIM-4 have been reported to be ligands for each other based on the binding of TIM-1-Ig to TIM-4 transfectants and vice versa (Meyers et al., 2005
); however, Biacore studies with highly purified protein showed the TIM-1:TIM-4 interaction was of very low affinity (Sizing et al., 2007
). In our assays of TIM-mediated phagocytosis, the apoptotic cell did not express TIM-1 or TIM-4, thereby excluding a TIM/TIM interaction. Some of the low level, universal binding of TIM proteins observed by ourselves and others (Cao et al., 2007
; Wilker et al., 2007
) may reflect the health of the cell and its level of exposure of PS. In addition, some apparent TIM-1/TIM-4 interactions could reflect a “bridge” with two TIM proteins binding to an exosome or membrane fragment via exposed PS. Such a bridge has been shown to be formed by MFG-E8 between an exosome and an apoptotic cell.
TIM-1 was also cloned as kidney injury molecule 1 (KIM-1) and is expressed minimally by healthy kidney but in post-ischemic kidney is highly unregulated on the luminal side of dedifferentiated tubule epithelial cells (Ichimura et al., 1998
). While the function of TIM-1 in recovery from ischemic kidney injury is not yet clear, our results show that TIM-1 on a renal cell carcinoma line mediates phagocytosis of apoptotic cells and suggests one of the functions of TIM-1 on dedifferentiated tubular epithelial cells may be recognition of dead cells after ischemic kidney injury and clearance of dead cells to reconstitute the tissue. In addition, TIM-1 ectodomain has been shown to be shed from the injured kidney (Han et al., 2002
) and may regulate the recognition of apoptotic cells either by opsonization or blockade. While TIM-1 can mediate phagocytosis in a large kidney cell, it may function on a small T cell more to receive signals via small exosomes or to sense the activation status of the T cell and the health of the APC and surrounding tissue.
administration of either TIM-4-Ig or TIM-1-Ig resulted in hyperproliferation of T cells and enhancement of T cell cytokine production (Meyers et al., 2005
). The administration of TIM-1 agonistic mAb enhanced T cell activation and proliferation and induced increased airway inflammation and blocked development of respiratory tolerance (Umetsu et al., 2005
). TIM-1 associates with T cell receptor and crosslinking with TIM-1 mAb leads to engagement of tyrosine signaling motifs in the cytoplasmic domain of TIM-1 and higher production of Th2 cytokines (Binne et al., 2007
; de Souza et al., 2005
). A common perplexing feature of in vivo
treatment with TIM mAbs or fusion proteins is the finding that at a time when T cells should have returned to quiescence, T cells taken from the treated animal and assayed in vitro
are still proliferating and producing cytokines. During a primary T cell response in vivo
, T cells expand exponentially and then 90% or more die by apoptosis and are phagocytosed. T cell activation leads to a rise of exposed PS on the surface of the T cell and this PS is associated with lipid rafts and the immunological synapse (Fischer et al., 2006
). While some in vitro
activated T cells express high levels of PS and progress to activation induced cell death, the majority express an intermediate level of PS, survive, and restore PS exposure to normal levels. TIM-mediated recognition of PS on activated T cells may have a role as T cells pass through this “near-death” experience. The extended T cell response caused by TIM blockade may be due to blockade of the phagocytosis of activated T cells exposing PS. Rapid removal of apoptotic cells by phagocytes is critical for the maintenance of tolerance and prevents inflammation and autoimmune responses against intracellular antigens released from the dying cells (Savill and Fadok, 2000
) (Wu et al., 2006
). Disruption of this process may explain the genetic association of the TIM gene family with auotoimmunity and asthma.
While the involvement of TIM-4 in phagocytes seems clear, the cytoplasmic domain of TIM-4 does not contain any tyrosine signaling motifs and the signaling pathway requires further study. In summary, TIM-1 and TIM-4 have unique structures that let them look death in the eye and give it a molecular kiss.