Abstract. Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (Ix) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34 ÷ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and teukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P-and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/Ix chimera to inhibit L-selectin/Ix binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L-and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L-and P-selectin interact with a domain located at the amino-terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L-and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P-and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L-selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ∼80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ∼80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.

Recombinant interferon-gamma (IFN-gamma) induced the expression of HLA-DR when added to the culture medium of HLA-DR- melanoma cell lines. In addition, IFN-gamma induced the expression of another class II antigen, HLA-DC, on a HLA-DR+ and -DC-melanoma cell line and to a lower level on a -DR- and -DC-melanoma line. IFN-gamma also enhanced the expression of HLA-ABC and beta 2-microglobulin, as well as HLA-DR on DR+ melanoma cells. In contrast, IFN-alpha gave no induction of expression of HLA-DR and DC on two DR- melanoma lines, while it did enhance the expression of HLA-ABC and of beta 2-microglobulin. The expression of 3 out of 6 melanoma-associated differentiation antigens was enhanced by IFN-gamma treatment. The modulation of antigens by IFN-gamma was both dose and time dependent. A minimum incubation time of 48 h was necessary for the appearance of HLA-DR on the two HLA-DR- melanoma lines, whereas HLA-ABC and beta 2-microglobulin were already increased after 24 h. A dose of 20 U/ml IFN-gamma started to induce the expression of HLA-DR and DC on melanoma cells GLL-19 and Me-43 and a plateau of maximum antigen expression was reached with 100 U/ml. Analyses of IFN-gamma-treated cells by flow microfluorometry showed a homogeneous distribution of increased staining intensity rather than the appearance of two cell populations. Immunoprecipitation experiments using detergent-solubilized 125I-labeled membrane proteins of IFN-gamma-treated melanoma cells and a monoclonal anti-HLA-DR antibody confirmed the presence of HLA-DR antigens. When IFN-gamma-treated cells were cultured without IFN the induced or enhanced expression of HLA antigens was reversible. Eight days after removal of IFN, the HLA-DR level was reduced by more than 90% and the level of HLA-ABC and beta 2-microglobulin by more than 50%. The demonstration of the ability of HLA-DR- melanoma cells to express HLA-DR after IFN-gamma treatment was extended to cells from other types of tumor such as gliomas, colon carcinomas and one cervical carcinoma cell line.

The expression on a significant number of thymocytes of idiotypic structures (Ti) restricted to HPB-ALL or Jurkat cells is demonstrated. As many as 2-4% of thymocytes were stained with anti-Ti HPB-ALL or anti-Ti Jurkat monoclonal antibodies, when analyzed by flow microfluorometry. Immunohistochemical localization studies performed on frozen thymus specimens of either fetal or pediatric origin indicated a scattered distribution of Ti-positive cells in both the cortex and the medulla. From lysates of 125I-labeled pediatric thymocytes, anti-Ti HPB-ALL and anti-Ti Jurkat monoclonal antibodies precipitated disulfide-linked heterodimers comparable to those precipitated from 125I-labeled HPB-ALL or Jurkat cells as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.