Identification of laminin domains involved in branching morphogenesis: Effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

Abstract

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

Characterization of α1, β1, and γ1 laminin subunits during rabbit fetal lung development

“…This is in contrast to the pattern of laminin staining we observed in the connective tissue of rabbit fetal lung tissue in which the intensity and frequency of laminin immunostaining increased with increasing gestational age. In agreement with our findings, Schuger et al (1991), using monoclonal antibodies that recognized regions in the a, p, and y subunit chains of laminin-1, reported that laminin immunostaining was present primarily in the basement membranes of airway epithelium, but some diffuse staining was observed in the extracellular matrix of the mesenchyme at an early stage of mouse lung development (day 12 of gestation).…”

“…Schuger et al (1991) demonstrated that monoclonal antibodies to specific regions of the laminin molecule interfered with the process of branching morphogenesis in murine lung explants. Laminin is also important in promoting the organotypic rearrangement of embryonic mouse lung single cell suspensions (Schuger et al, 1992).…”

Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin α1 chain, AL-2 with laminin β1 chain, and AL-4 with the coiled-coil domain of laminin β1 chain

“…By rotary shadowing and electron microscopy, the AL-4 was predicted to bind the end of the long arm, probably to the C-terminal globular domains of the LM α1 chain (Schuger et al, 1991). ELISA of AL-4 showed strong binding of EHS laminin, recombinant LM-111 as well as of the E8 fragment.…”

“…In order to study LM-111 or LM-121 it is therefore essential to use antibodies specific to the LM α1 chain. Rat monoclonal antibodies AL-1 and AL-4 have, based on rotary shadowing, been suggested to detect α1 chain (Schuger et al, 1991). AL-1 was predicted to detect a central part of the LM-111 trimer containing all three chains, whereas AL-4 was shown to detect the end of the long arm (Skubitz et al, 1988), which led Schuger et al 1991 to suggest that it reacts with the α1 chain.…”

“…AL-1 and AL-4 are nevertheless commercially marketed as α1-specific probes. AL-2, a commercially available antibody from the same series, is stated to react with the cross region of LM-111 (Skubitz et al, 1988;Schuger et al, 1991).…”