ABSTRACT
The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.
Subject(s)
Axons/physiology , Laminin/deficiency , Schwann Cells/cytology , Schwann Cells/physiology , Animals , Animals, Newborn , Axons/drug effects , Behavior, Animal/physiology , Cell Movement/genetics , Cells, Cultured , Central Nervous System/metabolism , Central Nervous System/pathology , Central Nervous System/ultrastructure , Demyelinating Diseases/genetics , Demyelinating Diseases/pathology , Immunohistochemistry/methods , In Vitro Techniques , Laminin/pharmacology , Laminin/physiology , Mice , Mice, Knockout/physiology , Microscopy, Electron, Transmission/methods , Muscle Spasticity/genetics , Nerve Regeneration/genetics , S100 Proteins/metabolism , Schwann Cells/drug effects , Sciatic Nerve/growth & development , Sciatic Nerve/pathology , Sciatic Nerve/ultrastructure , Sciatic Neuropathy/metabolism , Sciatic Neuropathy/pathology , Time FactorsABSTRACT
During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.
Subject(s)
Apoptosis , Cell Movement , Laminin/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Aflatoxin B1 , Animals , Antibodies, Monoclonal/immunology , Apoptosis/drug effects , Caseins/pharmacology , Cell Adhesion/drug effects , Endothelial Cells , Humans , Inflammation/chemically induced , Inflammation/immunology , Integrins/metabolism , Laminin/deficiency , Laminin/genetics , Laminin/pharmacology , Mice , Mice, Knockout , Neutrophils/drug effects , Peptide Fragments/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Serum AlbuminABSTRACT
The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.
Subject(s)
Laminin/genetics , Base Sequence , Cell Adhesion/physiology , Cell Line , Cell Movement/physiology , Chromatography, Affinity/methods , Cloning, Molecular , DNA Primers , DNA, Complementary , Humans , Laminin/isolation & purification , Laminin/physiology , Laminin/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructureABSTRACT
The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin alpha4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin alpha4 chain in microvessel growth and, in the absence of other laminin alpha chains, in the composition of endothelial basement membranes.
