Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa: effects on laminin-5 assembly, secretion, and deposition.

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G-->A) that results in in-frame skipping of exon 4 and synthesis of a mutated gamma2 polypeptide (gamma2Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma2t) disrupted in its alpha-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5 domain with a mild junctional epidermolysis bullosa phenotype.

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia.

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.

A homozygous mutation in the integrin alpha6 gene in junctional epidermolysis bullosa with pyloric atresia.

The alpha6 integrin subunit participates in the formation of both alpha6beta1 and alpha6beta4 laminin receptors, which have been reported to play an important role in cell adhesion and migration and in morphogenesis. In squamous epithelia, the alpha6beta4 heterodimer is the crucial component for the assembly and stability of hemidesmosomes. These anchoring structures are ultrastructurally abnormal in patients affected with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a recessively inherited blistering disease of skin and mucosae characterized by an altered immunoreactivity with antibodies specific to integrin alpha6beta4. In this report, we describe the first mutation in the alpha6 integrin gene in a PA-JEB patient presenting with generalized skin blistering, aplasia cutis, and defective expression of integrin alpha6beta4. The mutation (791delC) is a homozygous deletion of a single base (C) leading to a frameshift and a premature termination codon that results in a complete absence of alpha6 polypeptide. We also describe the DNA-based prenatal exclusion of the disease in this family at risk for recurrence of PA-JEB. Our results demonstrate that, despite the widespread distribution of the alpha6 integrin subunit, lack of expression of the alpha6 integrin chain is compatible with fetal development, and results in a phenotype indistinguishable from that caused by mutations in the beta4 chain, which is expressed in a more limited number of tissues.

Alpha 2,3-sialyl and alpha 1,3-fucosyltransferase-dependent synthesis of sialyl Lewis x, an essential oligosaccharide present on L-selectin counterreceptors, in cultured endothelial cells.

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L-selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of alpha 2,3-sialyl- (alpha 2,3-ST) and alpha 1,3-fucosyltransferases (alpha 1,3-FT) on N-acetyllactosamine. At least two of the several alpha 2,3-ST and four of the several alpha 1,3-FT are present in HUVEC. In functional assays both alpha 2,3-ST and alpha 1,3-FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the alpha 1,3-FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several alpha 2,3-ST and alpha 1,3-FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L-selectin counterreceptors.

Characterization of the E-selectin ligand on NK cells.

In this study we demonstrate that human CD56+CD16+/CD3- NK cells adhere to the E-selectin expressed by stimulated HUVEC in a sialidase- and Ca(2+)-dependent manner, and express a silylated Lex adhesion structure. We have characterized this sLe(x) epitope on NK cell in detail and show here that the sLe(x) on NK cells was not recognized by the CSLEX1 Ab, but was readily identified by two anti-di-sLe(x) Abs, KM-93 and FH-6. Furthermore, cleaving sialic acid with a sialidase treatment revealed a pool of Le(x) epitopes on the NK cells surface, providing further proof that NK cells express sLe(x) epitopes. Extensive protease treatments did not cleave the sLe(x) epitope from NK cells, which suggests that it could be linked to a lipid backbone. This di-sLe(x) was able to mediate adhesion to E-selectin, suggesting that it represents an essential part or is closely related to a selectin ligand on NK cells. We were also able to show that NK cells possess several alpha 2,3 sialyltransferases and alpha 1,3 or alpha 1,3/4 fucosyltransferases. These enzymes are crucial in the synthesis of sLe(x) epitopes on cell surfaces. Taken together, we provide evidence that NK cells have a di-sLe(x) oligosaccharide capable of adhesion to E-selectin, and NK cells have the machinery (i.e., relevant transferases) to generate these sialylated Lewis oligosaccharides.

Human NK cells expressing alpha 4 beta 1/beta 7 adhere to VCAM-1 without preactivation.

The authors demonstrate that resting CD56+/CD3- NK cell adhesion to the endothelial VCAM-1 is over three-fold higher than CD56-/CD3+ T-cell adhesion. T-cell, but not NK-cell adhesion, to VCAM-1 is enhanced significantly by stimulation. The expression of VCAM-1 receptor subunits alpha 4 and beta 1 on both effector cells remains unchanged upon stimulation. A subpopulation of NK cells, as well as of T cells, was found to express beta 7, whose expression was not altered upon stimulation. The authors conclude that the adhesive properties of the same receptor structures on these distinct cell populations are regulated in a different manner, according to the specific functions of the effector cells of the immune system.

Adhesion molecules involved in the interaction of LGL/NK cells and human endothelial cells stimulated with Salmonella bacteria.

Previously we showed that pretreatment of LGL/NK or HUVE cells with Salmonella bacteria augments the adhesion of LGL/NK cells to endothelium. Here we analyse the roles of HUVEC adhesion molecules VCAM-1, ICAM-1 and E-selectin, and the counter-receptors VLA-4, LFA-1 and SLex in the increase of LGL/NK adhesion to HUVEC, stimulated with Salmonella Minnesota mR595 bacteria, LPS or TNF-alpha. On Salmonella-stimulated HUVEC, VCAM-1 and ICAM-1 were the major binding structures involved, and their effect was additive in monoclonal antibody inhibition experiments. We could demonstrate the induction of both structures on cultured HUVEC after 24 h of Salmonella stimulation in flow cytometric analysis. For Salmonella-stimulated LGL/NK, the principal binding structure was LFA-1. Stimulation of LGL/NK cells did not alter the expression of the adhesion structures (subunits CD11a/CD18, CD49d/CD29), as determined by flow cytometric analysis, and thus the increased adherence is presumably produced by an increased avidity of the receptors on LGL/NK cells. Pretreatment of endothelium or lymphocytes with various stimuli, including Salmonella bacteria or LPS, leads to an activation state which provides for characteristic anchorage sites for the increased migration of LGL/NK cells towards the site of inflammation.

Effect of Salmonella bacteria on the interaction of human NK cells with endothelial cells.

Leukocyte-endothelial cell adhesion and its regulation are essential and complex initial aspects of lymphocyte migration. Various factors (IL-1, TNF-alpha, IFN-gamma etc.) have been shown to increase the endothelial adhesiveness for human lymphocytes, including natural killer cells (NK cells). In this work we have demonstrated that pretreatment of either the target endothelial cell monolayers or the binding LGL-cells with mR595 Salmonella Minnesota bacteria results in a substantial increase in the adhesiveness of LGL-cells to endothelial cells. The increase was more prominent when the endothelial cells were treated than when the adhering LGL-cells were similarly pretreated. The adhering cell population was significantly enriched with CD56 (Leu19) and CD16 positive cells, i.e. cells with NK cell phenotype, when the lymphocyte population was pretreated. However, the pretreatment of EC resulted in a non-specific increase in EC adhesiveness since the relative proportion of CD56+ (Leu19), CD16+ and CD3+ cells among the adhering cells did not significantly differ from the starting population. The bidirectional enhancement of adhesiveness of human NK cells to endothelium by mR595 Salmonella bacteria may be significant in the host defense responses against microbial infections.

Heat shock inhibits the cytotoxic action of TNF-alpha in tumor cells but does not alter its noncytotoxic actions in endothelial and adrenal cells.

We have previously demonstrated that a short heat treatment protects target cells from lysis by tumor necrosis factors (TNFs). Here we show that a similar heat treatment of human umbilical vein endothelial cells and human fetal adrenal cells does not alter noncytotoxic actions of TNF, suggesting that heat shock may specifically inhibit the cytotoxic action of TNF. To find clues to the mechanisms by which heat shock protects cells from TNF killing, its effects on TNF-alpha-TNF-receptor interactions, on the metabolism of the ligand, and on the expression of mRNAs for possible protective proteins were studied. The affinity of binding and the internalization of the ligand were slightly reduced after heat shock. These effects were, however, very vague and seen both in heat-responsive tumor cells and in endothelial and adrenal cells. Thus, it is unlikely that they could explain the heat-induced TNF resistance. Heat shock increased the expression of mRNAs for heat shock proteins (hsps) 27 and 70 in all the cells studied, but did not alter the expression of manganous superoxide dismutase (MnSOD) mRNA, which has previously been shown to play a crucial role in TNF resistance. Based on these results, we suggest that cells have multiple mechanisms to escape TNF-mediated lysis and that heat-induced protection from TNF killing may be mediated by hsps or other heat-inducible protective proteins, which act after receptor binding and protect cells from TNF-induced cellular damage without inhibiting the signal transduction mediating noncytotoxic effects of TNF.