Jack Y. Lee

SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

Dynamic alterations in chemokine gradients induce transendothelial shuttling of human T cells under physiologic shear conditions.

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies, yet the molecular effectors of this process are relatively uncharacterized. For extravasation, studies based predominantly on static transwell assays support a general model, whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However, this model of chemotaxis cannot readily reconcile how chemokines influence intravasation, as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus, to analyze how T cells integrate perivascular chemokine signals under physiologic flow, we developed a novel transwell‐based flow chamber allowing for real‐time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment, transmigrated T cells did not undergo retrograde transendothelial migration (retro‐TEM). However, when exposed to abluminal CXCL12, transmigrated T cells underwent striking retro‐TEM and re‐entered the flow stream. This CXCL12 fugetactic (chemorepellant) effect was concentration‐dependent, augmented by apical flow, blocked by antibodies to integrins, and reduced by AMD3100 in a dose‐dependent manner. Moreover, CXCL12‐induced retro‐TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream.

The mechanisms of up-regulation of dendritic cell activity by oxidative stress

Whereas DC have increasingly been recognized for their role in activating the inflammatory cascades during IRIs, the mechanisms by which oxidative stress enhances DC activation remain to be explored. We examined the role of oxidative stress on two important features of DC: T cell activation and trafficking. Bone marrow‐derived OS‐DC were compared with untreated DC. DC exposed to oxidative stress augmented allogeneic T cell proliferation and showed increased migration in a chemotaxis chamber. These results were confirmed by using hypoxanthine and xanthine oxidase as another inducer of oxidative stress. We used OT‐II and OT‐I mice to assess the effect of oxidative stress on DC activation of OVA‐specific CD4+ and CD8+ T cells, respectively. Oxidative stress increased DC capacity to promote OVA‐specific CD4+ T cell activity, demonstrated by an increase in their proliferation and production of IFN‐γ, IL‐6, and IL‐2 proinflammatory cytokines. Whereas oxidative stress increased the DC ability to stimulate IFN‐γ production by OVA‐specific CD8+ T cells, cellular proliferation and cytotoxicity were not affected. Compared with untreated DC, oxidative stress significantly reduced the capacity of DC to generate Tregs, which were restored by using anti‐IL‐6. With regard to DC trafficking, whereas oxidative stress increased DC expression of p‐Akt and p‐NF‐κB, targeting PI3Kγ and NF‐κB pathways abrogated the observed increase in DC migration. Our data propose novel insights on the activation of DC by oxidative stress and provide rationales for targeted therapies, which can potentially attenuate IRI.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.