Takashi Kei Kishimoto

Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.

Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators.

The localization of the adhesion protein L‐selectin in human neutrophils was determined by sub‐cellular fractionation and immunoelectron microscopy and compared with the localization of Mac‐1 (α m β z ) and alkaline phosphatase, the marker for secretory vesicles. L‐selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac‐1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeiov, H., et al. J. Clin. Invest. (1993) 92, 1467–1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet‐activating factor (PAF), or f‐Met‐Leu‐Phe (fMLP), induced parallel up‐regulation of the surface membrane content of alkaline phosphatase and Mac‐1 and down‐regulation of L‐selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L‐selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac‐1 were randomly distributed on the surface membrane of fMLP‐stimulated cells. These studies indicate that the transition of neutrophils from L‐selectin‐presenting cells to Mac‐l‐presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac‐1 and devoid of L‐selectin, into the plasma membrane. J. Leukoc. Biol. 56: 80–87; 1994.

CALMODULIN REGULATES L-SELECTIN ADHESION MOLECULE EXPRESSION AND FUNCTION THROUGH A PROTEASE-DEPENDENT MECHANISM

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.

Integrins, ICAMs, and Selectins: Role and Regulation of Adhesion Molecules in Neutrophil Recruitment to Inflammatory Sites

Publisher Summary This chapter discusses the key adhesion molecules involved in leukocyte trafficking to sites of inflammation, focuses on mechanisms to regulate adhesion molecule expression and function, and defines some of the steps involved in neutrophil–endothelial cell interactions. The chapter summarizes the progress made in testing anti-adhesion molecule monoclonal antibodies (MAbs) in animal models of inflammatory diseases. Neutrophils are the front line of defense and are rapidly mobilized and recruited to sites of tissue injury or infection. This efficient response requires that neutrophils gain access to virtually any tissue. In addition to traditional random screening of large chemical libraries, it may be possible to rationally design drugs based on known ligand structures, such as small carbohydrates for selectins or peptide-based antagonists. In addition to direct receptor antagonists, drugs that modulate adhesion molecule regulation or expression may also be effective.

Elevated Levels of Circulating Adhesion Molecules in IDDM Patients and in Subjects at Risk for IDDM

Serum levels of recently discovered circulating forms of adhesion molecules, ICAM-1 and L-selectin, were found to be elevated in IDDM patients and in subjects at risk for developing IDDM compared with 100 normal, nondiabetic blood donors. Both adhesion molecules were determined by sandwich ELISA. Serum concentrations of either clCAM-1 or cL-selectin were >2SD of normal mean in 10 of 14 recent-onset IDDM patients (P < 0.05). Serum levels of clCAM-1 and cL-selectin did not correlate. In first-degree relatives, elevated adhesion molecule levels were observed in the 6 ICA+ individuals and in the ICA− individuals all (n = 14) with a genetic risk of IDDM (sharing HLA-DR3 and/or -DR4− with the diabetic relative) but not in the HLA-DR3− and/or -DR4− relatives (n = 13). We conclude that elevated clCAM-1 and cL-selectin levels occur independently of ICA status and probably reflect ongoing immune processes in recent-onset IDDM patients and first-degree relatives at risk for IDDM.

ADAM17 but Not ADAM10 Mediates Tumor Necrosis Factor-α and L-Selectin Shedding from Leukocyte Membranes

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.

A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function

Lymphocyte function‐associated antigen(LFA)‐1/intercellular adhesion molecule (ICAM)‐1interactions mediate several important steps in the evolution of animmune response. LFA‐1 is normally expressed in a quiescent state onthe surface of leukocytes and interacts weakly with its ligands ICAM‐1,‐2, and ‐3. LFA‐1 activity may be regulated by receptor clustering andby increasing the affinity of LFA‐1 for its ligands. Affinitymodulation of LFA‐1 has been shown to occur via a conformational changein the LFA‐1 heterodimer that can be detected by using monoclonalantibody 24 (mAb24). We have recently described a small‐moleculeantagonist of LFA‐1, BIRT 377, that demonstrates selective in vitro andin vivo inhibition of LFA‐1/ICAM‐1‐mediated binding events. We nowdemonstrate that BIRT 377 blocks the induction of the mAb24 reporterepitope on LFA‐1 on the surface of SKW‐3 cells treated with variousagonists known to induce high‐affinity LFA‐1. These data imply thatBIRT 377 exerts its inhibitory effects by preventing up‐regulation ofLFA‐1 to its high‐affinity conformation.

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils.

The signaling factors that direct the rapid shedding of L‐selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N‐formylmethionyl‐leucyl‐phenylalanine (fMLP)‐induced L‐selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP‐ and phorbol 12‐myristate 13‐acetate (PMA)‐induced L‐selectin shedding; however, these inhibitors did not affect fMLP‐induced up‐regulation of Mac‐1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP‐induced L‐selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant‐induced L‐selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L‐selectin shedding from neutrophils. J. Leukoc. Biol. 67: 415–422; 2000.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.