Expression of adhesion molecules in synovia of patients with treatment-resistant lyme arthritis.

The expression of adhesion molecules in synovium in patients with Lyme arthritis is surely critical in the control of Borrelia burgdorferi infection but may also have pathologic consequences. For example, molecular mimicry between a dominant T-cell epitope of B. burgdorferi outer surface protein A and an adhesion molecule, human lymphocyte function-associated antigen 1 (LFA-1), has been implicated in the pathogenesis of treatment-resistant Lyme arthritis. Using immunohistochemical methods, we examined synovial samples for expression of adhesion molecules in 29 patients with treatment-resistant Lyme arthritis and in 15 patients with rheumatoid arthritis or chronic inflammatory monoarthritis. In Lyme arthritis synovia, endothelial cells showed intense expression of P-selectin and vascular adhesion protein-1 (VAP-1). Expression of LFA-1 was also intense on infiltrating cells, particularly in lymphoid aggregates, and intercellular adhesion molecule-1 (ICAM-1) was markedly expressed on synovial lining and endothelial and infiltrating cells. Moderate expression of vascular cell adhesion molecule-1 (VCAM-1) was seen on synovial lining and endothelial cells, and mild expression of its ligand, very late antigen-4, was apparent in perivascular lymphoid infiltrates. Except for lesser expression of VCAM-1 in Lyme synovia, the levels of expression of these adhesion molecules were similar in the three patient groups. We conclude that certain adhesion molecules, including ICAM-1 and LFA-1, are expressed intensely in the synovia of patients with Lyme arthritis. Upregulation of LFA-1 on lymphocytes in this lesion may be critical in the pathogenesis of treatment-resistant Lyme arthritis.

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

Cloning of the mucosal addressin MAdCAM-1 from human brain: identification of novel alternatively spliced transcripts.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed selectively on high endothelial venules (HEV) and lamina propria venules, directs lymphocyte traffic by binding the lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1, alpha 4 beta 7). Full-length DNA encoding human MAdCAM-1 was obtained by combining sequences from an expressed sequence tag (EST) identified in an early stage human brain cDNA library, a polymerase chain reaction-derived clone, and a MAdCAM-1 genomic clone. The deduced amino acid sequence revealed an 18 amino acid signal peptide, two N-terminal immunoglobulin (Ig)-like domains conserved (59-65%) in sequence with those of the mouse homologue, an 86 amino acid mucin-like region rich in serine-threonine residues, a 20 amino acid transmembrane domain and a 43 amino acid charged cytoplasmic domain. No counterpart to the third IgA-like domain of mouse MAdCAM-1 was present; however, the serine-threonine-rich mucin domain was extended as two distinguishable major and minor mucin regions unrelated to the mouse domain. The major domain is formed from six tandem repeats of an eight amino acid sequence having the MUC-2-related consensus DTTSPEP/SP. Human MAdCAM-1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas and brain. Alternatively spliced MAdCAM-1 variants were identified that lack parts of the second Ig domain and all or part of the major mucin domain, indicating that the function of this vascular addressin is regulated by extensive modifications to its multi-domain structure.

Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29).

Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.

Immunopathogenesis of vernal keratoconjunctivitis.

We have analyzed the in situ distribution of immune cells in the conjunctival biopsy specimens obtained from patients with active vernal keratoconjunctivitis (VKC). We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies. Our data point to a complex immunopathogenesis of the disease. Distinct components involved in IgE-mediated immune mechanisms, as well as humoral and cell mediated immune mechanisms were detected in the conjunctival tissues. In addition, we investigated the presence and distribution of adhesion molecules. In the normal conjunctiva, intercellular adhesion molecule-1 (ICAM-1) was expressed only on the vascular endothelium, lymphocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) on epithelial and stromal mononuclear cells, and very late activation antigen-4 (VLA-4) on a few stromal mononuclear cells. Endothelial leukocyte adhesion molecule-1 (ELAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression was not detected. In VKC a marked increase of all these antigens was observed. Strong ICAM-1 expression was induced on the basal epithelial cells, and vascular endothelium. Furthermore, about 30% of the stromal mononuclear cells expressed ICAM-1. LFA-1 and ICAM-3 were expressed on the majority of infiltrating mononuclear cells. VLA-4 expression was noted on about 25% of the stromal mononuclear cells. ELAM-1 and VCAM-1 were induced on the vascular endothelial cells. Our results suggests that increased expression of adhesion molecules in VKC promotes the recruitment of inflammatory cells through blood vessels and the cell interaction between lymphocytes and antigen presenting cells, among lymphocytes, as well as between lymphocytes and epithelial cells.

Isolation and characterisation of a hyaluronan binding protein, hyaluronectin, from human placenta and its colocalisation with hyaluronan.

Hyaluronan (HA) is a major component of the extracellular matrix and is known to influence cell behaviour and to play a role in angiogenesis, morphogenesis and tissue remodelling, although little is known concerning the regulation of these effects. Until now its detection in the placenta has been by indirect methods, which has led to conflicting conclusions as to its distribution and hence its role. Hyaluronectin (HN) is one of a group of proteins with HA binding ability which may regulate the effects of HA. Although nervous tissue HN has been partly characterised with regard to its distribution, structure and biochemistry, little is known about the mesenchymal isoform and its distribution in placenta has not previously been reported. Using specific probes we have characterised the distribution of HA and HN in human placental tissue. At all stages of development studied (8, 10, 12, 30 and 38 wk gestation) HA and HN were unequivocally colocalised, being distributed in the extracellular matrix of stromal tissue of placental villi, chorioallantoic membranes and umbilical cord. Particularly strong immunoreactivity was observed in the villous stroma immediately adjacent to fibrinoid depositions at sites of denudation of the trophoblast layer. Extraction and characterisation of the HN from placental villi have revealed 4 major glycoproteins of 47, 52, 57 and 67 kDa, this being a different pattern and smaller molecular range than observed for the nervous tissue form. This is the first direct demonstration of the presence of HA and HN in the placenta and identifies an abundant new source of mesenchymal HN. The functions of mesenchymal HN are unknown but may include ion exchange, immunosuppression and regulation of the effects of HA in such roles as maintenance of tissue architecture, cell migration and angiogenesis.

Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan.

Our laboratory has previously characterized and purified the hyaluronan receptor by hyaluronan affinity chromatography of rat liver endothelial cells. We have now isolated the receptor from whole rat liver and have obtained sufficient quantities for amino acid sequence analysis. Four peptides of various lengths were obtained from affinity-purified receptor and found to have identity with rat intercellular adhesion molecule-1. This glycoprotein is normally expressed in low amounts on the endothelial cells, but is up-regulated in inflamed and malignant tissues, and mediates cell-cell adhesion as a ligand for lymphocyte function-associated antigen-1 and the macrophage-associated Mac-1. The affinity of intercellular adhesion molecule-1 for hyaluronan is likely to have important implications for cell adhesion in normal and in disease states such as inflammation, atherosclerosis, and cancer.

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.

A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways.

Evidence for the existence of hyaluronectin-binding proteins in the plasma membranes.

This report documents for the first time the existence of specific binding proteins for hyaluronic acid binding protein (hyaluronectin) in the plasma membranes of normal and transformed cells. Firstly, we showed the specific binding of hyaluronic acid binding protein to the cell surface of normal rat heart fibroblasts (NRHF) by saturation and competition methods using 125I-labeled hyaluronic acid binding protein and calculated the binding dissociation constant (0.43 x 10(-13) M). In order to identify hyaluronectin-binding protein on the cell surface, plasma membranes isolated from rat brain, liver and fibrosarcoma were separated by SDS-PAGE and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred membrane proteins with 125I-labeled hyaluronectin in the presence of non-ionic as well as ionic detergents revealed two prominent bands of approximate molecular mass of 37 kDa and 40 kDa in brain, liver and fibrosarcoma. The specificity of the binding [125I]hyaluronectin to 37-kDa and 40-kDa membrane proteins was further confirmed, as the intensity of the bands was reduced in the presence of a 20-fold excess of unlabeled hyaluronectin. We discuss our observations on hyaluronectin-binding membrane proteins in the context of hyaluronectin-mediated cellular functions.

Isolation and characterization of the soluble and membrane-bound porcine CD44 molecules.

Porcine CD44 was identified by the cross-reactive anti-human CD44 monoclonal antibody (mAb) Hermes-1. Material which inhibited the Hermes-1 mAb binding to porcine lymphocytes was found in porcine intestinal efferent lymph and demonstrated to be a soluble form of porcine CD44. A protocol using non-affinity techniques was established to isolate this material from porcine lymph. The isolated soluble CD44 molecule consists of a single peptide with an apparent MW of about 50,000, containing the Hermes-1 epitope. The soluble and membrane-bound forms of porcine CD44 were also affinity purified and characterized. The soluble CD44 preparation comprised a major component of 48,000-70,000 MW peptide and a minor band of 120,000 MW. The membrane-bound CD44 preparation contained a 90,000 MW major and a 120,000 MW minor component. Both the major components of the soluble and membrane-bound forms of porcine CD44 contained N-linked glycosylation. The 48,000-70,000 MW component of the soluble CD44 molecules consisted of two subsets discriminated by their binding capacity for lentil lectin and a slight difference in molecular weights. Soluble CD44 molecules were also identified in the sera of sheep, goats, horses, dogs and humans. Like the porcine counterpart, these molecules could be enriched by anion-exchange chromatography, suggesting that they have similar biochemical properties.

CD44 expression on murine tissues.

CD44 is a cell surface glycoprotein found on lymphoid and epithelial cells. Its primary function on lymphocytes and macrophages is to mediate interaction with endothelium, while its function on epithelial cells is not known. The protein has many different forms, generated by alternative mRNA splicing and by post-translational modification, which may mediate different functions. During previous work on murine lung tumor cells, mAb 133-13A was isolated and shown to recognize a surface glycoprotein, P100, of 90-100 x 10(3) M(r). Amino acid sequence analysis of purified P100 indicates that it is CD44. Since few data exist to indicate which forms of CD44 are present in different normal tissues, mAb 133-13A was used to analyze CD44 expression in mouse tissue. Quantitative data on the distribution of CD44(P100) in mice show that spleen, thymus, liver, intestine, uterus and choroid of the eye are major sites of expression. In addition, epithelia of adrenals, esophagus and trachea are CD44(P100) positive. Previous work on human cell lines has implicated a high molecular mass (130-160 x 10(3) M(r)) form of the glycoprotein as the form expressed in epithelial cells and carcinomas. Isolation of CD44 proteins from lymphoid tissues in the mouse indicate that, as in human lymphoid tissue, the low molecular mass form (80-90 x 10(3) M(r)) is predominately expressed. These data show that both small (approximately 81 x 10(3) M(r)) and large forms of the glycoprotein are expressed in basal epithelia of esophagus and trachea and in salivary gland, while only the small form is expressed in epithelium of the adrenal cortex and in the murine lung and mammary carcinomas studied. While these data cannot distinguish between specific splice variants, they show that the large forms of CD44 are minor components in normal tissue and seem to be found only in basal epithelium. The CD44 of low M(r) found in epithelial tissues is probably associated with lymphoid cell types in the tissues.

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.