Regulation of Endothelial Cell Adhesion Molecule Expression with Antisense Oligonucleotides

C. Frank Bennett; Stanley T. Crooke    Department of Molecular Pharmacology ISIS Pharmaceuticals Carlsbad, California 92008

I Leukocyte Migration

Leukocyte emigration through postcapillary venules is an active process requiring an orchestrated interaction of a number of proteins with their respective ligands (Fig. 1). Best characterized is the migration of neutrophils through endothelium in response to inflammatory stimuli. Neutrophil emigration can be categorized into at least three distinct steps, reversible adhesion, leukocyte activation, and stable binding (Butcher, 1991). Intravital microscopy was used more than 100 years ago to describe these steps (Cohnheim, 1889). In the past few years, some of the molecular interactions which occur during this process have been characterized. The first step which is observed is rolling of leukocytes along endothelium. Rolling of leukocytes on the surface of vascular endothelium requires transient attachment to and detachment from the endothelium. For the process to be efficient, the ligands which mediate this transient attachment should not exhibit high affinities for their binding proteins. Rolling has been demonstrated to be due to increased expression of P-selectin and E-selectin on the vascular endothelium, both of which interact with carbohydrates, such as sialyl Lex on circulating neutrophils (Lawrence and Springer, 1991). In addition, L-selectin on the neutrophil may also participate in the rolling process.

Neutrophils undergo an “activation” event in response to soluble stimuli such as chemotactic peptide, leukotriene B4, platelet-activating factor, or interleukin 8, cell matrix-associated cytokines or through interactions with proteins expressed on endothelial cells (Butcher, 1991; Tanaka et al., 1993). On activation, dramatic changes in the shape of neutrophils can be observed with the cells becoming flattened and exhibiting pseudopodia. In addition, the avidity of the neutrophil proteins leukocyte function- associated antigen-1 (LFA-1) and Mac-1 for their ligand intercellular adhesion molecule 1 (ICAM-1) increases, resulting in stable binding to the endothelium (Dustin, 1990). T cells have also been demonstrated to undergo activation resulting in enhanced binding of LFA-1 to ICAM-1 (Dustin and Springer, 1989). This activation of binding is thought to be due to phosphorylation of the β-subunit of either LFA-1 or Mac-1 (Hibbs et al., 1991). In most instances, transendothelial migration requires CD18 and ICAM-1 (Furie et al., 1991; Oppenheimer-Marks et al., 1991). Although vascular cell adhesion molecule 1 (VCAM-1) may play an important role in the initial binding of some leukocyte types to the endothelium, VCAM-1 does not appear to play a role in transendothelial migration (Oppenheimer- Marks et al., 1991; Ebisawa et al., 1992). This conclusion is supported by the finding that VCAM-1 is expressed on the apical surface of endothelial cells, while ICAM-1 is present on both the apical and basal surface of endothelial cells as well as expressed at intracellular junctions.

II Endothelial-Leukocyte Adhesion Molecules

There has been much progress in defining and characterizing the macro molecules responsible for the adhesive interactions between leukocytes and vascular endothelium and characterizing their role in normal homeostasis. Members of at least three multigene families are involved as well as unique carbohydrate structures and additional poorly characterized molecules (Table I). In addition, there are several proteins identified by use of monoclonal antibodies which function in lymphocyte adherence and migration into specialized lymphoid structures which have currently unknown primary sequences (Picker, 1992). A brief discussion regarding the better-characterized proteins involved in leukocyte migration follows.

A Immunoglobulin Family

The immunoglobulin gene superfamily is a large family of proteins which have in common one or more 90 to 100 amino acid domains, characterized by two antiparallel β-pleated sheets usually held together by a disulfide bond (Williams and Barclay, 1988). Although members of this family are functionally diverse, most are cell-surface proteins involved in recognition of other molecules. This recognition can be either homophilic or heterophilic. The genetic loci coding for members of the immunoglobulin gene superfamily are found on multiple chromosomes. Three members of the immunoglobulin gene family are expressed on endothelial cells and are involved in leukocyte emigration and activation, ICAM-1, ICAM-2, and VCAM-1, with a fourth member, ICAM-3, expressed on leukocytes.

ICAM-1 was identified by use of a monoclonal antibody which recognized a heavily glycosylated cell-surface protein of 90 to 110 kDa, distinct from LFA-1, expressed at low levels on unstimulated endothelial cells and circulating leukocytes (Dustin et al., 1986; Rothlein et al., 1986). Expression of ICAM-1 was found to be markedly elevated in response to interleukin 1 (IL1), tumor necrosis factor (TNF), interferon-γ (IFN’γ), and bacterial lipopolysaccharide (LPS) in a wide variety of cells including endothelial cells, keratinocytes, fibroblasts, and astrocytes.

ICAM-1 has five extracellular immunoglobulin-like domains, a 27 amino acid signal sequence, a putative 24 amino acid transmembrane domain, and a short 28 amino acid cytoplasmic domain (Simmons et al., 1988; Staunton et al., 1988). The gene for ICAM-1 is located on chromosome 19 and consists of seven exons, with each of the immunoglobulin domains coded for by separate exons. The transmembrane domain, cytoplasmic domain, and the 3’-untranslated region are all contained in the last exon (Voraberger et al., 1991). The mRNA encoding ICAM-1 is approximately 3.0 kb in length, containing a long 1400 nucleotide 3’-untranslated region. The 3’-untranslated region of ICAM-1 mRNA contains three repeats of AUUUA sequence which have been proposed to confer instability to mRNAs containing such sequences (Caput et al., 1986). Several proteins have been identified that bind selectively to these AUUUA sequences; however, their function in mRNA stability is still unknown (Bohjanen et al., 1991; Vakalopoulou et al., 1991). Human ICAM-1 exhibits 50% homology to mouse and rat ICAM-1 (Siu et al., 1989; Horley et al., 1989; Kita et al., 1992).

ICAM-1 binds to circulating leukocytes via interactions with the β2- integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) (Marlin and Springer, 1987; Diamond et al., 1990). Binding to LFA-1 has been mapped to the first immunoglobulin domain while binding to Mac-1 occurs at a distinct site on the third immunoglobulin domain (Staunton et al., 1990; Diamond et al., 1990, 1991). Studies suggest that binding to Mac-1 may be regulated in part by the glycosylation state of domain 3, with deglycosylated forms exhibiting more pronounced binding. In contrast, binding to LFA-1 does not appear to be influenced by glycosylation (Diamond et al., 1991). ICAM-1 has also been shown to bind CD43, also known as sialophorin or leukosialin...