Legionella pneumophila replicates in macrophages in a host-derived phagosome, termed the Legionella-containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with all L. pneumophila strains tested and in amoebae, natural hosts for L. pneumophila. It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM.

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CD99-Like 2 (CD99L2) is a Type I glycoprotein expressed on leukocytes and endothelial cells as well as other cell types. It is related to CD99, although it shows only 38% sequence identity. CD99L2 has been shown to play a role in leukocyte extravasation in mice under various inflammatory conditions using anti-CD99L2 antibodies and, in one case by targeted deletion of CD99L2. We report here studies on an independently made CD99L2 “knockout mouse” that extend our knowledge of the role of CD99L2 in inflammation. CD99L2 deficiency did not affect the total or relative numbers of circulating leukocyte subsets, red blood cells, or platelets. Neither did CD99L2 deficiency affect the expression of ICAM-1, PECAM, or CD99 on endothelial cells. Mice lacking CD99L2 had a defective inflammatory response in the thioglycollate peritonitis model with a greater than 80% block in neutrophil infiltration and a nearly complete block in monocyte emigration into the peritoneal cavity measured 16 hours after the inflammatory challenge. The mice will be a useful resource to study the role of CD99L2 in various acute and chronic inflammatory diseases.

CD99-like 2 (CD99L2 [L2]) is a highly glycosylated 52-kDa type 1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function-blocking Ab significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation. However, the role of L2 in inflammation has only been studied in mice. Furthermore, the mechanism by which it regulates TEM is not known. To study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Our data show that like PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Inhibiting L2 using Ab blockade or genetic knockdown significantly reduces transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.

Transendothelial Migration (TEM) is a crucial step in the inflammatory response as it is the process in which leukocytes leave the bloodstream to enter the inflamed tissues. Adhesion/signaling molecules such as PECAM and CD99 are important for the initiation and completion of TEM. We and others have shown that CD99‐like 2 (L2), a highly glycosylated 52 kD type‐1 membrane protein that is the only molecule in the genome related to CD99 is also important for TEM. However, L2 is different enough from CD99 (60% larger and with only 38% sequence identity) that it is not clear whether it regulates TEM by the same mechanism as CD99. Furthermore, all of the published studies on L2 have been performed in mice. The role of L2 in human cells is not known. In order to study the mechanism used by L2 and the relevance to human inflammation, we studied the role of L2 on human cells in vitro. Our data show that similar to PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes; treatment with proinflammatory cytokines changes neither its surface expression nor its distribution. Blocking L2 using antibodies significantly reduces transmigration of human neutrophils and monocytes across human endothelial cells. Blockade of either leukocytes or endothelial cells blocks equivalently to blocking both, consistent with a homophilic mechanism of interaction. Furthermore, knockdown of L2 using shRNAs dose‐dependently reduces TEM of neutrophils and monocytes across endothelial cells. TEM is restored by re‐expression of L2. Our data also show that L2 regulates a step in TEM after PECAM and before CD99. Consistent with this, preliminary data suggest that L2 can promote TEM by engaging either the signaling pathway used by PECAM or the one used by CD99. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to the site of TEM. Ongoing studies are focused on identifying unique signaling pathways (if any) used by L2 to promote TEM. Support or Funding Information T32‐5T32AI007476‐19 R37‐HL064774 F31‐HL131355‐01 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.