Arnoud Sonnenberg

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain.

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino‐terminal sequence is in good agreement with the published amino‐terminal sequence of purified beta 4. The extracellular domain contains five potential N‐linked glycosylation sites and four cysteine‐rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.

Antibody to integrin α6 subunit specifically inhibits cell-binding to laminin fragment 8

Abstract A large number of cell lines which attach and spread on laminin show a comparable binding either to both laminin fragments P1 and E8 or exclusively to E8. Adhesion to fragment E8 was with one exception completely inhibited by a monoclonal antibody to the α6 integrin subunit, indicating that VLA-6 or a related structure is the major cellular receptor for laminin. It is not involved in fragment P1 adhesion. Synthetic peptides possessing RGD or YIGSR sequences were without inhibitory activity for α6-mediated adhesion to fragment E8.

Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1

A single RGD‐containing sequence present within an epidermal growth factor‐like repeat of the short arms of laminin is shown by peptide inhibition to block integrin receptors recognizing a latent cell‐binding site of laminin. Based on proteolysis data it is proposed that masking occurs by folding of the globular domain IVa over the cell‐binding site in the adjacent rod‐like structures of laminin A chain.

Suppression of mouse melanoma metastasis by EA-1, a monoclonal antibody specific for alpha 6 integrins

The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block the adhesion of T lymphocyte progenitors to mouse thymic endothelium. Here we show that the mAb EA-1 recognizes the alpha 6 chain of alpha 6 beta 1 and alpha 6 beta 4 integrins. Both molecules are present at a high level on the luminal and basolateral side of vascular endothelium and alpha 6 beta 1 integrin is expressed on the highly metastatic cell lines B16/129 (melanoma) and KLN-205 (carcinoma). These lung specific tumors bind preferentially to lung frozen sections, and EA-1 blocked this interaction in vitro. Moreover, mAb EA-1 inhibited experimental metastasis to the lung of B16/129 cells injected intravenously. Metastasis in vivo was blocked when the antibody was injected into mice before or simultaneously with the melanoma cells, as well as when melanoma cells were precoated with EA-1 before injection. We suggest that alpha 6 integrins play a dual role in the metastatic process, mediating the adhesion of tumor cells to the luminal surface of the endothelium and the adhesion to laminin in the subendothelial extracellular matrix during extravasation. Despite the fact that alpha 6 integrins are laminin receptors, EA-1 did not interfere with melanoma cell binding to laminin fragments. Our antibody EA-1 may therefore recognize a binding domain on alpha 6 integrins of a novel ligand involved in cell-cell interaction.

Isolation of α6β1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Abstract Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OVCAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the α6β1 integrin by immunoprecipitation with specific antibodies against either the α6 or the β1 subunit. The α6β1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the αIIbβ3 integrin from platelets. Furthermore, in immunofluorescence staining, the α6β1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the α6β1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin α3β1. When α3β1 was removed from lysates of OVCAR-4 cells by preclearing with an α3-specific monoclonal antibody, α6β1 was able to bind to human laminin as well. Integrin α6β1 on platelets which do not express α3β1 binds directly to human laminin. These results indicate that both α3β1 and α6β1 can act as receptors for human laminin and may interfere by steric hindrance. The α6β4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.

Determination of proteinase 3—α1-antitrypsin complexes in inflammatory fluids

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is α1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3—α1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3—α1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)—α1AT complexes. Thus, in vivo α1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

A novel specificity of anticytoplasmic autoantibodies directed against eosinophil peroxidase.

Vasculitis associated anticytoplasmic autoantibodies (ANCA) are directed against enzymes in the granules of both neutrophils and monocytes. These autoantibodies can be detected by indirect immunofluorescence technique (IIFT) using ethanol‐fixed cytospins. We here report the identification of a novel specificity of autoantibodies, present in the sera of eight patients, that reacted only with eosinophils in the IIFT. By immunoprecipitation and ELISA experiments it was shown that the autoantibodies in these sera were directed against eosinophil peroxidase (EPO). There was no apparent influence on initial substrate conversion rate, but reduced plateau levels suggested increased inactivation of the enzyme in the course of the peroxidase reaction. Flow cytometry studies demonstrated the presence of EPO on the surface of primed eosinophils. Anti‐EPO sera and purified anti‐EPO immunoglobulins significantly increased the release of reactive oxygen species from primed eosinophils. The patients with anti‐EPO antibodies suffered from clinically diverse disorders, with more or less generalized manifestations involving the kidneys, blood vessels, lungs and/or joints.

ANCA related antigens.

Sera from patients with Wegener’s Granulomatosis (WG) contain antineutrophil cytoplasmic autoantibodies (ANCA), that produce a typical granular cytoplasmic staining in indirect immunofluorescence on ethanol fixed cytospins of normal donor neutrophils. The antibodies recognize a 29kD serine protease, located in the primary granules of myeloid leucocytes. A number of sera submitted for routine ANCA testing produced an atypical (pen-) nuclear staining in the standard ANCA immunofluorescence test (ANCA IIFT). Immunoprecipitation from radiolabeled extract from neutrophil azurophilic granules, showed that these atypical staining sera often specifically recognized myeloperoxidase (MPO). However, in a minority of these sera we found antibodies against leucocyte elastase. The presence of these antibodies was confirmed by antigen-catching ELISA in which monoclonal antibodies to the 29 kD antigen, MPO and elastase were used as catching antibodies. Anti-elastase antibodies were detected in sera from a heterogenous group of 10 patients with renal and/or respiratory tract manifestations and systemic features. In the sera of two patients who were treated with propylthiouracil for hyperthyroidism we found antibodies against the 29 kD antigen and antibodies against elastase. ANA was negative. Clinically both patients suffered from fever, arthralgia and high ESR. One patient had necrotizing leucocytoclastic vasculitis and the other patient had conjunctivitis and scleritis suggesting a vasculitic disease. After cessation of therapy their clinical condition improved markedly and the antibody titer in one case disappeared and in the other case fell from 1:1024 to 1:128. Recently we found antibodies directed against eosinophil peroxidase (EPO). In the IIFT, sera from three patients showed reactivity only with eosinophils. In an immunoprecipitation using radiolabeled eosinophil lysate these sera precipitated bands at Mr 58 kD and 14 kD in a 1 : 1 stoichometry, i.e. the same ratio as the control polyclonal antibody rabbit anti-EPO. Specificity for EPO was further established by immunoprecipitation using radiolabeled purified MPO and EPO. In a solid phase ELISA reactivity was tested against purified MPO and EPO coated to microtiter plates. In this assay the three sera showed only reactivity with EPO. Absence of cross reactivity of anti-EPO with anti-MPO was further demonstrated by immunoprecipitation cross inhibition studies with excess of unlabeled antigen. The three patients showed different clinical manifestations: one had cyclic neutropenia, one unclassified systemic vasculitis and one had unclassified systemic disease with relapsning aphtic ulcers in the mouth and throat, possibly Morbus BehCet. We finally report a patient with rapidly progressive glomerulonephritis and serum-antibodies against MPO, elastase and lactoferrin. There were no signs of systemic disease. Histologically this patient was classified as idiopathic crescentic glomerulonephritis. We conclude that anti-elastase, anti-EPO and antilactoferrin further extend the range of anti-myeloid granule antibodies and may occur either as single specificities or in combination with other ANCA. The limited number of patients in whom these antibodies have thus far been detected prevents conclusions on their diagnostic value or pathogenetic importance.