Mark H. Siegelman

Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion.

The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium, and is governed, in part, by the expression of adhesion molecules on both cell types. Recently, we have described a novel primary adhesion interaction between the structurally activated form of the adhesion molecule CD44 on lymphocytes and its major ligand hyaluronan on endothelial cells under physiologic laminar flow conditions, and have proposed that this interaction functions in an extravasation pathway for lymphocytes in vascular beds at sites of inflammation. While the regulation of activated CD44 on leukocytes has been characterized in depth, regulation of hyaluronate (HA) on endothelial cells has not been extensively studied. Here we demonstrate that the expression of HA on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFalpha and IL-1beta, as well as bacterial lipopolysaccharide. In addition, this inducibility appears strikingly restricted to endothelial cells derived from microvascular, but not large vessel, sources. The elevated HA levels thus induced result in increased CD44-dependent adhesive interactions in both nonstatic shear and laminar flow adhesion assays. Changes in mRNA levels for the described HA synthetic and degradative enzymes were not found, suggesting other more complex mechanisms of regulation. Together, these data add to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and help to further our understanding of the potential physiologic roles of the CD44/HA interaction; i.e., local cytokine production within inflamed vascular beds may enhance surface hyaluronan expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells.

The mouse lymph node homing receptor is identical with the lymphocyte cell surface marker Ly-22: Role of the EGF domain in endothelial binding

The lymph node homing receptor core polypeptide (mLHRc) is composed of a tandem collection of domains: a lectin domain, an epidermal growth factor (EGF) domain, and two repeats common in complement regulatory proteins. Here we demonstrate localization of mLHRc to chromosome 1, the portion syntenic with chromosome 1 in man. This locus is inseparable in mouse strains from the murine lymphocyte cell surface marker Ly-22. The data indicate that Ly-22 is an allelic determinant on the LHR resulting from a single amino acid interchange within the EGF domain. Cross-blocking experiments demonstrate that anti-Ly-22 and MEL-14 recognize independent epitopes and that Ly-22 is distinct from the carbohydrate binding region. Application of anti-Ly-22 in the in vitro binding assay shows inhibition of binding of lymphocytes to high endothelial venules (HEVs). The localization of the Ly-22 epitope in this novel chimeric protein suggests direct participation of the EGF domain in the adhesion of lymphocytes to HEV.

More than the sum of the parts: cooperation between leukocyte adhesion receptors during extravasation

Over the past decade, it has become clear that the specificity and targeted regulation of leukocyte trafficking result from the sequential and frequently overlapping functions of various adhesive receptors. Adhesion between the leukocyte and the vessel wall begins with repeated transient interactions that allow the leukocyte to “roll” across the endothelium at a rate lower than that of blood flow. Rolling is attributed to the interaction between carbohydrate ligands and either members of the selectin family (1, 2) or CD44 (3, 4) and is followed by firm adhesion mediated by receptors of a different class, the leukocyte integrins. These integrins require activation-induced avidity increases to bind their ligands, generally members of the immunoglobulin gene superfamily. The primary relevant integrins expressed by leukocytes contain either α4 (the heterodimers α4β1 and α4β7) or β2, also known as CD18, which heterodimerizes with any of four distinct α chains to form LFA-1, Mac-1, p150,95, and αdβ2 (5). The combination of adhesion receptor expression and microenvironmental influences encountered by leukocytes as they traverse the endothelium controls integrin activation and determines the leukocyte subsets recruited to a particular site and the kinetics with which this recruitment occurs. Loss of any of the adhesion molecules in this scheme could result in reduced extravasation of particular leukocyte subsets at sites of inflammation. Early studies of leukocyte adhesion molecule deficiency 1 disease (LADI), a genetic deficiency in which affected patients are subject to recurrent bacterial and fungal infections and have impaired wound healing, showed mutations in CD18. It is perhaps fitting that the manuscript by Forlow et al. in a recent issue of the JCI (6) focuses in part on the same molecule. Mice lacking all three selectins, the β2 integrins, ICAM-1, or various combinations of these adhesion receptors have been generated with varying outcomes in induced inflammatory models, but generally these strains remain healthy under standard laboratory conditions (7, 8). The earliest indication of a spontaneous inflammatory defect resulting from adhesion receptor gene targeting was seen in mice doubly deficient for E- and P-selectin. Similar to the phenotype reported in the present study, the abnormalities in these mice consist primarily of excoriative skin lesions associated with bacterial colonization in the head and neck (9, 10). Rolling interactions are severely compromised in E/P-selectin–deficient mice, which fail to induce normal inflammatory responses to peritoneal infection with Streptococcus pneumoniae. These studies clearly established rolling as a prerequisite for firm adhesion. The second instance of a spontaneous and severe inflammatory condition was identified in CD18-deficient mice, again consisting primarily of mucocutaneous lesions (11). Nearly all mice of this genotype develop a similar progressive ulcerative dermatitis and are highly susceptible to inoculated S. pneumoniae. Intravital microscopy revealed that firm adhesion but not rolling is markedly impaired in these mice.

Lymphocyte Homing Receptors, Ubiquitin, and Cell Surface Proteins

The immune system, unlike most organ systems that are consolidated in one anatomic location, is dispersed over an entire organism. It exists as circulating elements in the blood, through which it gains access to nearly all body tissues, and as innumerable lymphoid aggregates throughout the body. Therefore, the immune system is placed under a special constraint, which is managed by substituting extensive cell-cell recognition and interactive events as a strategy for organization. Of course, these recognition events are not required for the imposed proximity of cells in architecturally localized organ systems. This layer of complexity must then be superimposed on the central function of the immune system, self-nonself-recognition and appropriate responses to nonself-encounters. These functions are largely executed by the major element of the immune system, the lymphocyte.

Structural Characterization of a Murine Lymphocyte Homing Receptor Suggests a Ubiquitinated Branched-Chain Glycoprotein

Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino-termini, one of which corresponds precisely to the amino-terminus of ubiquitin. This observation extends the province of this extraordinarily conserved polypeptide to the cell surface, and leads to a proposed model of the receptor complex as a ubiquitinated branched-chain glycoprotein. Functional binding of lymphocytes to high endothelial venules (HEV) requires the accessibility of the ubiquitinated region of the receptor, suggesting a possible central role for ubiquitin in cell-cell interaction and adhesion.