Sulfhydryl Regulation of L-Selectin Shedding: Phenylarsine Oxide Promotes Activation-Independent L-Selectin Shedding from Leukocytes 1 Teresa A. Bennett,* Bruce S. Edwards,* Larry A. Sklar,* ² and Snezna Rogelj 2 * The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhy- dryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-{D,L-[2-(hydroxyami- nocarbonyl)-methyl]-4-methylpentanoyl}-L-3-(tert-butyl)-alanyl-L-alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hy- pothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis. The Journal of Immunology, 2000, 164: 4120 – 4129. T he recruitment of neutrophils to inflammatory sites and lymphocyte trafficking among blood, lymphoid, and non- lymphoid tissues involve a regulated sequence of adhe- sive interactions among adhesion molecules of the selectin, mucin, integrin, and ICAM families (1, 2). At the receptor level, expres- sion, conformation, and proteolytic cleavage are among the mech- anisms by which cellular adhesive interactions can be controlled. L-selectin is expressed on the majority of leukocytes, including peripheral blood T and B lymphocytes, neutrophils, eosinophils, basophils, monocytes, NK cells, and some subpopulations of thy- mocytes (3). Under flow-induced high shear forces, L-selectin plays a critical role in initiating the interactions of these cells with the activated endothelium at an inflammatory site (4, 5). In addi- tion, L-selectin is thought to amplify the inflammatory process by permitting adherent neutrophils to recruit additional neutrophils (6–8). Lymphocytes require L-selectin for trafficking across the high endothelial venules into the peripheral lymph nodes (2) and to discriminate between the Th1- vs. Th2-type cytokine-producing T cells (9). L-selectin also plays a decisive role in the development of other neutrophil- and lymphocyte-mediated pathological pro- cesses, including ischemia-reperfusion injury, septic shock, graft rejection, autoimmune diseases, the metastasis of lymphoid tumors (10 –13), and HIV-induced CD4 + cell depletion (14). L-selectin shedding is one important aspect of the normal phys- iologic regulation of L-selectin adhesive function. Cell surface ex- pression of this adhesion molecule is characteristically down-mod- ulated in response to cell activation (3, 15, 16). This proteolytic release from neutrophils inhibits subsequent L-selectin-dependent interactions with other neutrophils and endothelial cells at inflam- matory sites (17, 18). Lymphocyte L-selectin is shed in response to activation by PMA (15), bacterial superantigens (19), or, like neu- trophil L-selectin, by the treatment of cells with Abs to L-selectin (20, 21). L-selectin loss results in profound changes in T cell re- circulation pathways (22), and studies with L-selectin-deficient mice have revealed a dramatic (70 –90%) reduction in the number of lymphocytes in peripheral lymph nodes (22, 23). The released, soluble L-selectin retains binding capacity and may function as an adhesive buffer by preventing leukocyte adhesion at sites of sub- acute inflammation (24). Increased levels of plasma L-selectin are found in several disease states, including AIDS (25). L-selectin shedding is the result of a proteolytic cleavage close to its transmembrane domain, conducted by a constitutively active membrane metalloprotease (26, 27), recently shown to be identical with TNF- converting enzyme (28). Several groups, including ours, have shown that hydroxamic acid-based inhibitors of matrix metalloproteases, such as (N-{D,L-[2-(hydroxyaminocarbonyl)- methyl]-4-methylpentanoyl}-L-3-(tert-butyl)-alanyl-L-alanine,2- aminoethyl amide (TAPI-2), 3 inhibit the L-selectin sheddase and *Department of Pathology, Division of Cytometry, Cancer Research Facility, Uni- versity of New Mexico School of Medicine, Albuquerque, NM 87131; and ² National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, NM 87545 Received for publication October 29, 1999. Accepted for publication February 1, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant RO1RR14175 (to L.A.S.) and the SCOR Program, Project PPG HL56384 (Principle Investigator: M. Lipscomb). 2 Address correspondence and reprint requests to Dr. Snezna Rogelj, Jones Annex Room 315, Department of Biology, New Mexico Institute of Mining and Technology, Socorro, NM 87801. E-mail address: snezna@nmt.edu 3 Abbreviations used in this paper: TAPI-2, (N-{D,L-[2-(hydroxyaminocarbonyl)- methyl]-4-methylpentanoyl}-L-3-(tert-butyl)-alanyl-L-alanine, 2-aminoethyl amide; PAO, phenylarsine oxide; PDI, protein disulfide isomerase; DMP, 2,3-dimercapto- Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00